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1.
Anticancer Res ; 40(7): 3781-3792, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32620617

RESUMO

BACKGROUND/AIM: Canine B-cell lymphoma represents a useful in vivo model for human diffuse large B-cell lymphoma (DLBCL). Pan-Bromodomain and extra-terminal (BET) inhibition targeting BRD2/3/4 and selective inhibition of BRD4, as well as spleen tyrosine kinase (SYK) inhibition, are currently evaluated as haematologic cancer therapy. Herein, we characterized the differences in the biologic response of isoform-specific or pan-BET inhibition alone or in combination with SYK inhibition. MATERIALS AND METHODS: I-BET151 (pan-inhibitor) and AZD5153 (BRD4 inhibitor) were combined with Entospletinib (SYK inhibitor) and comparatively analysed in the canine DLBCL cell line CLBL-1. Dose- and time-dependent cellular responses were analysed by cell number, metabolic activity, apoptosis/necrosis, and cell morphology. The synergistic potential was evaluated through the Bliss independence model. RESULTS: I-BET151 and AZD5153 showed significant dose- and time-dependent inhibitory effects. Adding Entospletinib to I-BET151 or AZD5153 had no additional synergistic effects. CONCLUSION: Entospletinib did not enhance the inhibitory effects of the pan- or isoform-specific BET.


Assuntos
Indazóis/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Isoformas de Proteínas/antagonistas & inibidores , Pirazinas/farmacologia , Animais , Linhagem Celular Tumoral , Cães , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Linfoma Difuso de Grandes Células B/metabolismo , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Quinase Syk/metabolismo
2.
PLoS One ; 15(7): e0236119, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667929

RESUMO

The alcohol-abuse drug disulfiram has antitumor effects against diverse cancer types via inhibition of the ubiquitin-proteasome protein nuclear protein localization protein 4 (NPL4). However, the antitumor effects of NPL4 and disulfiram in clear cell renal cell carcinoma (ccRCC) are unclear. Here, we evaluated the therapeutic potential of targeting the ubiquitin-proteasome pathway using disulfiram and RNA interference and investigated the mechanisms underlying disulfiram in ccRCC. According to data from The Cancer Genome Atlas, NPL4 mRNA expression was significantly upregulated in clinical ccRCC samples compared with that in normal kidney samples, and patients with high NPL4 expression had poor overall survival compared with patients with low NPL4 expression. Disulfiram and NPL4 siRNA inhibited ccRCC cell proliferation in vitro, and disulfiram inhibited ccRCC tumor growth in a xenograft model. Synergistic antiproliferative effects were observed for combination treatment with disulfiram and sunitinib in vitro and in vivo. In RCC cells from mice treated with disulfiram and/or sunitinib, several genes associated with serine biosynthesis and aldose reductase were downregulated in cells treated with disulfiram or sunitinib alone and further downregulated in cells treated with both disulfiram and sunitinib. These findings provided insights into the mechanisms of disulfiram and suggested novel therapeutic strategies for RCC treatment.


Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Dissulfiram/farmacologia , Reposicionamento de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Renais/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Acetaldeído Desidrogenases/farmacologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Prognóstico , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
PLoS Pathog ; 16(6): e1008538, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32544190

RESUMO

Zika virus (ZIKV) infects pregnant women and causes devastating congenital zika syndrome (CZS). How the virus is vertically transmitted to the fetus and induces neuronal loss remains unclear. We previously reported that Pellino (Peli)1, an E3 ubiquitin ligase, promotes p38MAPK activation in microglia and induction of lethal encephalitis by facilitating the replication of West Nile virus (WNV), a closely related flavivirus. Here, we found that Peli1 expression was induced on ZIKV-infected human monocytic cells, peripheral blood mononuclear cells, human first-trimester placental trophoblasts, and neural stem cell (hNSC)s. Peli1 mediates ZIKV cell attachment, entry and viral translation and its expression is confined to the endoplasmic reticulum. Moreover, Peli1 mediated inflammatory cytokine and chemokine responses and induced cell death in placental trophoblasts and hNSCs. ZIKV-infected pregnant mice lacking Peli1 signaling had reduced placental inflammation and tissue damage, which resulted in attenuated congenital abnormalities. Smaducin-6, a membrane-tethered Smad6-derived peptide, blocked Peli1-mediated NF-κB activation but did not have direct effects on ZIKV infection. Smaducin-6 reduced inflammatory responses and cell death in placental trophoblasts and hNSCs, and diminished placental inflammation and damage, leading to attenuated congenital malformations in mice. Collectively, our results reveal a novel role of Peli1 in flavivirus pathogenesis and suggest that Peli1 promotes ZIKV vertical transmission and neuronal loss by mediating inflammatory cytokine responses and induction of cell death. Our results also identify Smaducin-6 as a potential therapeutic candidate for treatment of CZS.


Assuntos
Síndrome de Guillain-Barré , Proteínas Nucleares/antagonistas & inibidores , Peptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Infecção por Zika virus , Zika virus/metabolismo , Animais , Linhagem Celular , Feminino , Síndrome de Guillain-Barré/tratamento farmacológico , Síndrome de Guillain-Barré/genética , Síndrome de Guillain-Barré/metabolismo , Síndrome de Guillain-Barré/patologia , Humanos , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais/genética , Trofoblastos/metabolismo , Trofoblastos/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Zika virus/genética , Infecção por Zika virus/tratamento farmacológico , Infecção por Zika virus/genética , Infecção por Zika virus/metabolismo , Infecção por Zika virus/patologia
4.
Nucleic Acids Res ; 48(11): 6019-6031, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32379321

RESUMO

ALT tumor cells often contain abundant DNA damage foci at telomeres and rely on the alternative lengthening of telomeres (ALT) mechanism to maintain their telomeres. How the telomere chromatin is regulated and maintained in these cells remains largely unknown. In this study, we present evidence that heterochromatin protein 1 binding protein 3 (HP1BP3) can localize to telomeres and is particularly enriched on telomeres in ALT cells. HP1BP3 inhibition led to preferential growth inhibition of ALT cells, which was accompanied by telomere chromatin decompaction, increased presence of C-circles, more pronounced ALT-associated phenotypes and elongated telomeres. Furthermore, HP1BP3 appeared to participate in regulating telomere histone H3K9me3 epigenetic marks. Taken together, our data suggest that HP1BP3 functions on telomeres to maintain telomere chromatin and represents a novel target for inhibiting ALT cancer cells.


Assuntos
Proliferação de Células , Montagem e Desmontagem da Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Telômero/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Eucromatina/genética , Eucromatina/metabolismo , Técnicas de Silenciamento de Genes , Heterocromatina/genética , Código das Histonas , Histonas/química , Humanos , Metilação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Multimerização Proteica , Homeostase do Telômero
5.
J Med Chem ; 63(10): 5477-5487, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32367723

RESUMO

Protein arginine methyltransferase 6 (PRMT6) plays important roles in several biological processes associated with multiple cancers. Well-characterized potent, selective, and cell-active PRMT6 inhibitors are invaluable tools for testing biological and therapeutic hypotheses. Although there are several known reversible PRMT6 inhibitors, covalent PRMT6 inhibitors have not been reported. Based on a cocrystal structure of PRMT6-MS023 (a type I PRMT inhibitor), we discovered the first potent and cell-active irreversible PRMT6 inhibitor, 4 (MS117). The covalent binding mode of compound 4 to PRMT6 was confirmed by mass spectrometry and kinetic studies and by a cocrystal structure. Compound 4 did not covalently modify other closely related PRMTs, potently inhibited PRMT6 in cells, and was selective for PRMT6 over other methyltransferases. We also developed two structurally similar control compounds, 5 (MS167) and 7 (MS168). We provide these valuable chemical tools to the scientific community for further studying PRMT6 physiological and pathophysiological functions.


Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Estrutura Secundária de Proteína , Proteína-Arginina N-Metiltransferases/metabolismo
6.
J Med Chem ; 63(10): 5139-5158, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32315177

RESUMO

AIMP2-DX2, a splicing variant of AIMP2, is up-regulated in lung cancer, possesses oncogenic activity, and results in tumorigenesis. Specifically inhibiting the interaction between AIMP2-DX2 and HSP70 to suppress AIMP2-DX2-dependent cancers with small molecules is considered a promising avenue for cancer therapeutics. Optimization of hit BC-DXI-04 (IC50 = 40.1 µM) provided new potent sulfonamide based AIMP2-DX2 inhibitors. Among these, BC-DXI-843 showed improved inhibition against AIMP2-DX2 (IC50 = 0.92 µM) with more than 100-fold selectivity over AIMP2 in a luciferase assay. Several binding assays indicated that this compound effectively induces cancer cell apoptosis by specifically interrupting the interaction between DX2 and HSP70, which leads to the degradation of DX2 via Siah1-mediated ubiquitination. More importantly, BC-DXI-843 demonstrated in vivo efficacy in a tumor xenograft mouse model (H460 cells) at a dosage of 50 mg/kg, suggesting it as a promising lead for development of novel therapeutics targeting AIMP2-DX2 in lung cancer.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Desenvolvimento de Medicamentos/métodos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Células A549 , Animais , Antineoplásicos/farmacologia , Sulfonatos de Arila/síntese química , Sulfonatos de Arila/metabolismo , Sulfonatos de Arila/farmacologia , Células CHO , Cricetinae , Cricetulus , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
J Med Chem ; 63(8): 3908-3914, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32208684

RESUMO

Aminoacyl-tRNA synthetase interacting multifunctional proteins (AIMPs) have recently been considered novel therapeutic targets in several cancers. In this publication we report the development of novel 2-aminophenylpyrimidines as new AIMP2-DX2 inhibitors. In particular, aminophenylpyrimidine 3 not only exhibited promising in vitro and in vivo potency but also exerted selective inhibition of H460 and A549 cells and AIMP2-DX2 rather than WI-26 cells and AIMP2. Aminophenylpyrimidine 3 offers possible therapeutic potential in the treatment of lung cancer.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Células A549 , Aminoacil-tRNA Sintetases/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Nucleares/metabolismo , Pirimidinas/química , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
8.
Cancer Res ; 80(8): 1693-1706, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32054769

RESUMO

A significant therapeutic challenge for patients with cancer is resistance to chemotherapies such as taxanes. Overexpression of LIN9, a transcriptional regulator of cell-cycle progression, occurs in 65% of patients with triple-negative breast cancer (TNBC), a disease commonly treated with these drugs. Here, we report that LIN9 is further elevated with acquisition of taxane resistance. Inhibiting LIN9 genetically or by suppressing its expression with a global BET inhibitor restored taxane sensitivity by inducing mitotic progression errors and apoptosis. While sustained LIN9 is necessary to maintain taxane resistance, there are no inhibitors that directly repress its function. Hence, we sought to discover a druggable downstream transcriptional target of LIN9. Using a computational approach, we identified NIMA-related kinase 2 (NEK2), a regulator of centrosome separation that is also elevated in taxane-resistant cells. High expression of NEK2 was predictive of low survival rates in patients who had residual disease following treatment with taxanes plus an anthracycline, suggesting a role for this kinase in modulating taxane sensitivity. Like LIN9, genetic or pharmacologic blockade of NEK2 activity in the presence of paclitaxel synergistically induced mitotic abnormalities in nearly 100% of cells and completely restored sensitivity to paclitaxel, in vitro. In addition, suppressing NEK2 activity with two distinct small molecules potentiated taxane response in multiple in vivo models of TNBC, including a patient-derived xenograft, without inducing toxicity. These data demonstrate that the LIN9/NEK2 pathway is a therapeutically targetable mediator of taxane resistance that can be leveraged to improve response to this core chemotherapy. SIGNIFICANCE: Resistance to chemotherapy is a major hurdle for treating patients with cancer. Combining NEK2 inhibitors with taxanes may be a viable approach for improving patient outcomes by enhancing mitotic defects induced by taxanes alone.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Quinases Relacionadas a NIMA/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Paclitaxel/farmacologia , Taxoides/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas Supressoras de Tumor/antagonistas & inibidores , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Senescência Celular , Centrossomo/enzimologia , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Xenoenxertos , Humanos , Mitose/genética , Quinases Relacionadas a NIMA/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Paclitaxel/administração & dosagem , Taxa de Sobrevida , Taxoides/administração & dosagem , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Ensaio Tumoral de Célula-Tronco , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima
9.
Am J Respir Cell Mol Biol ; 62(6): 732-746, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32048876

RESUMO

Pulmonary vasoconstriction resulting from intermittent hypoxia (IH) contributes to pulmonary hypertension (pHTN) in patients with sleep apnea (SA), although the mechanisms involved remain poorly understood. Based on prior studies in patients with SA and animal models of SA, the objective of this study was to evaluate the role of PKCß and mitochondrial reactive oxygen species (mitoROS) in mediating enhanced pulmonary vasoconstrictor reactivity after IH. We hypothesized that PKCß mediates vasoconstriction through interaction with the scaffolding protein PICK1 (protein interacting with C kinase 1), activation of mitochondrial ATP-sensitive potassium channels (mitoKATP), and stimulated production of mitoROS. We further hypothesized that this signaling axis mediates enhanced vasoconstriction and pHTN after IH. Rats were exposed to IH or sham conditions (7 h/d, 4 wk). Chronic oral administration of the antioxidant Tempol or the PKCß inhibitor LY-333531 abolished IH-induced increases in right ventricular systolic pressure and right ventricular hypertrophy. Furthermore, scavengers of O2- or mitoROS prevented enhanced PKCß-dependent vasoconstrictor reactivity to endothelin-1 in pulmonary arteries from IH rats. In addition, this PKCß/mitoROS signaling pathway could be stimulated by the PKC activator PMA in pulmonary arteries from control rats, and in both rat and human pulmonary arterial smooth muscle cells. These responses to PMA were attenuated by inhibition of mitoKATP or PICK1. Subcellular fractionation and proximity ligation assays further demonstrated that PKCß acutely translocates to mitochondria upon stimulation and associates with PICK1. We conclude that a PKCß/mitoROS signaling axis contributes to enhanced vasoconstriction and pHTN after IH. Furthermore, PKCß mediates pulmonary vasoconstriction through interaction with PICK1, activation of mitoKATP, and subsequent mitoROS generation.


Assuntos
Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Mitocôndrias/fisiologia , Proteína Quinase C beta/fisiologia , Artéria Pulmonar/fisiopatologia , Vasoconstrição/fisiologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Células Cultivadas , Óxidos N-Cíclicos/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Depuradores de Radicais Livres/farmacologia , Humanos , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Hipóxia/enzimologia , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Canais de Potássio/metabolismo , Mapeamento de Interação de Proteínas , Artéria Pulmonar/enzimologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Síndromes da Apneia do Sono/fisiopatologia , Marcadores de Spin , Acetato de Tetradecanoilforbol/farmacologia
10.
J Mol Biol ; 432(7): 2204-2216, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32087201

RESUMO

Tools for actively targeted DNA demethylation are required to increase our knowledge about regulation and specific functions of this important epigenetic modification. DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5-meC), which may promote its replication-dependent dilution and/or active removal through base excision repair (BER). However, it is still unclear whether oxidized derivatives of 5-meC are simply DNA demethylation intermediates or rather epigenetic marks on their own. Unlike animals, plants have evolved enzymes that directly excise 5-meC without previous modification. In this work, we have fused the catalytic domain of Arabidopsis ROS1 5-meC DNA glycosylase to a CRISPR-associated null-nuclease (dCas9) and analyzed its capacity for targeted reactivation of methylation-silenced genes, in comparison to other dCas9-effectors. We found that dCas9-ROS1, but not dCas9-TET1, is able to reactivate methylation-silenced genes and induce partial demethylation in a replication-independent manner. We also found that reactivation induced by dCas9-ROS1, as well as that achieved by two different CRISPR-based chromatin effectors (dCas9-VP160 and dCas9-p300), generally decreases with methylation density. Our results suggest that plant 5-meC DNA glycosylases are a valuable addition to the CRISPR-based toolbox for epigenetic editing.


Assuntos
5-Metilcitosina/química , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes , Proteínas Nucleares/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Epigênese Genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Ativação Transcricional
11.
Nat Commun ; 11(1): 740, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029739

RESUMO

Primary and acquired drug resistance imposes a major threat to achieving optimized clinical outcomes during cancer treatment. Aberrant changes in epigenetic modifications are closely involved in drug resistance of tumor cells. Using BET inhibitor (BETi) resistant leukemia cells as a model system, we demonstrated herein that genome-wide enhancer remodeling played a pivotal role in driving therapeutic resistance via compensational re-expression of pro-survival genes. Capitalizing on the CRISPR interference technology, we identified the second intron of IncRNA, PVT1, as a unique bona fide gained enhancer that restored MYC transcription independent of BRD4 recruitment in leukemia. A combined BETi and CDK7 inhibitor treatment abolished MYC transcription by impeding RNAPII loading without affecting PVT1-mediated chromatin looping at the MYC locus in BETi-resistant leukemia cells. Together, our findings have established the feasibility of targeting enhancer plasticity to overcome drug resistance associated with epigenetic therapies.


Assuntos
Leucemia Experimental/tratamento farmacológico , Leucemia Experimental/genética , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Elementos Facilitadores Genéticos , Feminino , Genes myc/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Humanos , Células Jurkat , Células K562 , Leucemia Experimental/metabolismo , Camundongos , Modelos Genéticos , Fenilenodiaminas/administração & dosagem , Pirimidinas/administração & dosagem , RNA Polimerase II/metabolismo , RNA Longo não Codificante/genética
12.
Life Sci ; 248: 117454, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32088211

RESUMO

AIMS: Dihydroartemisinin (DHA) is currently considered as the promising cancer therapeutic drug. In this study, we aimed to investigate the anti-proliferative and anti-metastasis effects of DHA. MAIN METHODS: Utilizing breast cancer cells MCF-7, MDA-MB-231 and BT549, cell proliferation, migration and invasion were detected. RT-qPCR was performed to detect CIZ1, TGF-ß1 and Snail expression, and the interactions of these related molecules were analyzed by GeneMANIA database. Western blot detected CIZ1, TGF-ß1/Smads signaling and Snail expression in DHA-treated cells, in TGFß1-induced cells with enhanced metastatic capacity, and in cells treated with DHA plus TGFß1/TGFß1 inhibitor SD-208. KEY FINDINGS: Results indicated DHA inhibited breast cancer cell proliferation and migration, with more potent effects compared with that of artemisinin. RT-qPCR and Western blot showed DHA inhibited CIZ1, TGF-ß1 and Snail expression, and these molecules were shown to have protein-protein interactions by bioinformatics. Furthermore, TGFß1-treatment enhanced MCF-7 migration and invasion, and CIZ1, TGF-ß1/Smads signaling and snail activities; DHA, SD-208, combination of DHA and SD-208 reversed these conditions, preliminarily proving the cascade regulation between TGF-ß1 signaling and CIZ1. MCF-7 xenografts model demonstrated the inhibition of DHA on tumor burden, and its mechanisms and well-tolerance in vivo; combination of DHA and SD-208 tried by us for the first time showed better treatment effects, but possible liver impairment made its use still keep cautious. SIGNIFICANCE: DHA treatment inhibits the proliferation and metastasis of breast cancer, through suppressing TGF-ß1/Smad signaling and CIZ1, suggesting the promising potential of DHA as a well-tolerated antitumor TGF-ß1 pathway inhibitor.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Artemisininas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/genética , Fator de Crescimento Transformador beta1/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal , Feminino , Humanos , Metástase Linfática , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Pteridinas/farmacologia , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fatores de Transcrição da Família Snail/antagonistas & inibidores , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
PLoS One ; 15(1): e0220348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935221

RESUMO

In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated.


Assuntos
Acetiltransferases/genética , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Células Epiteliais/enzimologia , Fibroblastos/enzimologia , Acetiltransferases/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Proliferação de Células , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Quebra Cromossômica , Segregação de Cromossomos , Anormalidades Craniofaciais/enzimologia , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Ectromelia/enzimologia , Ectromelia/genética , Ectromelia/patologia , Células Epiteliais/patologia , Fibroblastos/patologia , Expressão Gênica , Humanos , Hipertelorismo/enzimologia , Hipertelorismo/genética , Hipertelorismo/patologia , Camundongos , Mitose , Modelos Biológicos , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
14.
Nucleic Acids Res ; 48(4): 1800-1810, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31930333

RESUMO

Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.


Assuntos
Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Transcrição Genética , Estrogênios/metabolismo , Hormônios Esteroides Gonadais/genética , Histona Acetiltransferases/genética , Humanos , Conformação Molecular , Proteínas Nucleares/antagonistas & inibidores , Ligação Proteica/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Análise de Célula Única
15.
Endocrinology ; 161(2)2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31875887

RESUMO

The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway has cell-specific functions. Suppressors of cytokine signaling (SOCS) proteins are negative-feedback regulators of JAK-STAT signaling. STAT5 plays a significant role in adipocyte development and function, and bromodomain and extraterminal (BET) proteins may be involved in STAT5 transcriptional activity. We treated 3T3-L1 adipocytes with the BET inhibitor JQ1 and observed that growth hormone (GH)-induced expression of 2 STAT5 target genes from the SOCS family, Socs3 and Cish, were inversely regulated (increased and decreased, respectively) by BET inhibition. Chromatin immunoprecipitation analyses revealed that changes in STAT5 binding did not correlate with gene expression changes. GH promoted the recruitment of the BET protein BRD2 to the Cish, but not Socs3, promoter. JQ1 treatment ablated this effect as well as the GH-induced binding of ribonucleic acid polymerase II (RNA Pol II) to the Cish transcription start site. BRD2 knockdown also suppressed GH induction of Cish, further supporting the role of BRD2 in Cish transcriptional activation. In contrast, JQ1 increased the binding of activated Pol II to the Socs3 coding region, suggesting enhanced messenger RNA (mRNA) elongation. Our finding that JQ1 transiently reduced the interaction between the positive transcription elongation factor (P-TEFb) and its inhibitor hexamethylene bis-acetamide inducible 1 (HEXIM1) is consistent with a previously described off-target effect of JQ1, whereby P-TEFb becomes more available to be recruited by genes that do not depend on BET proteins for activating transcription. These results demonstrate substantially different transcriptional regulation of Socs3 and Cish and suggest distinct roles in adipocytes for these 2 closely related proteins.


Assuntos
Adipócitos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Azepinas , Camundongos , Fator B de Elongação Transcricional Positiva/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Triazóis
16.
Biomed Pharmacother ; 121: 109368, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31707348

RESUMO

Hypertension is an essential regulator of cardiac injury and remodeling. However, the pathogenesis that contributes to cardiac hypertrophy remains to be fully explored. BRD4, as a bromodomain and extra-terminal (BET) family member, plays an important role in critical biological processes. In the study, our results showed that BRD4 expression was up-regulated in human and mouse hypertrophied hearts, and importantly these effects were modulated by reactive oxygen species (ROS) generation. In angiotensin II (Ang II)-treated cardiomyocytes, BRD4 decrease markedly blunted the prohypertrophic effect, which was further promoted by the combinational treatment of ROS scavenger (N-acetyl-cysteine, NAC). In addition, NAC pre-treatment markedly elevated the anti-fibrotic role of BRD4 suppression in Ang II-incubated cardiomyocytes by repressing transforming growth factor ß1 (TGF-ß1)/SMADs signaling pathway. NAC combined with BRD4 reduction further alleviated inflammation and oxidative stress in Ang II-exposed cardiomyocytes, which was partly through inhibiting nuclear factor-κB (NF-κB) signaling and improving nuclear erythroid factor 2-related factor 2 (Nrf-2)/heme oxygenase-1 (HO-1) pathway, respectively. Furthermore, the in vivo results confirmed the protective effects of BRD4 suppression on mice against aortic banding (AB)-induced cardiac hypertrophy, as evidenced by the reduced cross sectional area and fibrotic area using H&E and Masson trichrome staining. What's more, the degree of cardiac hypertrophy (ANP and BNP), the expression of pro-fibrotic genes (TGF-ß1, Collagen I, Collagen III and CTGF), the levels of inflammation and oxidative stress were all significantly attenuated by the blockage of BRD4 in AB-operated mice. Taken together, repressing BRD4 expression was found to confer a protective effect against experimental cardiac hypertrophy in mice, demonstrating its potential as an effective therapeutic target for pathological cardiac hypertrophy.


Assuntos
Cardiomegalia/metabolismo , Fibrose/metabolismo , Inflamação/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Angiotensina II/farmacologia , Animais , Cardiomegalia/tratamento farmacológico , Linhagem Celular , Fibrose/tratamento farmacológico , Coração/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Humanos , Inflamação/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima/efeitos dos fármacos
17.
Breast Cancer Res ; 21(1): 138, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31805991

RESUMO

BACKGROUND: The tumor suppressor actions of hexamethylene bis-acetamide (HMBA)-inducible protein 1 (HEXIM1) in the breast, prostate, melanomas, and AML have been reported by our group and others. Increased HEXIM1 expression caused differentiation and inhibited proliferation and metastasis of cancer cells. Historically, HEXIM1 has been experimentally induced with the hybrid polar compound HMBA, but HMBA is a poor clinical candidate due to lack of a known target, poor pharmacological properties, and unfavorable ADMETox characteristics. Thus, HEXIM1 induction is an intriguing therapeutic approach to cancer treatment, but requires better chemical tools than HMBA. METHODS: We identified and verified KDM5B as a target of HEXIM1 inducers using a chemical proteomics approach, biotin-NeutrAvidin pull-down assays, surface plasmon resonance, and molecular docking. The regulation of HEXIM1 by KDM5B and KDM5B inhibitors was assessed using chromatin immunoprecipitation assays, RT-PCR, western blotting, and depletion of KDM5B with shRNAs. The regulation of breast cancer cell phenotype by KDM5B inhibitors was assessed using western blots, differentiation assays, proliferation assays, and a mouse model of breast cancer metastasis. The relative role of HEXIM1 in the action of KDM5B inhibitors was determined by depleting HEXIM1 using shRNAs followed by western blots, differentiation assays, and proliferation assays. RESULTS: We have identified a highly druggable target, KDM5B, which is inhibited by small molecule inducers of HEXIM1. RNAi knockdown of KDM5B induced HEXIM1 expression, thus validating the specific negative regulation of tumor suppressor HEXIM1 by the H3K4me3/2 demethylase KDM5B. Known inhibitors of KDM5B were also able to induce HEXIM1 expression, inhibit cell proliferation, induce differentiation, potentiate sensitivity to cancer chemotherapy, and inhibit breast tumor metastasis. CONCLUSION: HMBA and 4a1 induce HEXIM1 expression by inhibiting KDM5B. Upregulation of HEXIM1 expression levels plays a critical role in the inhibition of proliferation of breast cancer cells using KDM5B inhibitors. Based on the novel molecular scaffolds that we identified which more potently induced HEXIM1 expression and data in support that KDM5B is a target of these compounds, we have opened up new lead discovery and optimization directions.


Assuntos
Regulação Neoplásica da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Biomarcadores Tumorais , Neoplasias da Mama/etiologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/química , Estimativa de Kaplan-Meier , Modelos Moleculares , Estadiamento de Neoplasias , Proteínas Nucleares/química , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas de Ligação a RNA/química , Recidiva , Proteínas Repressoras/química , Relação Estrutura-Atividade , Fatores de Transcrição/química
18.
Phys Chem Chem Phys ; 21(45): 25276-25289, 2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31701109

RESUMO

As a member of the bromodomain and extra terminal domain (BET) protein family, bromodomain-containing protein 4 (BRD4) is an epigenetic reader and can recognize acetylated lysine residues in histones. BRD4 has been regarded as an essential drug target for cancers, inflammatory diseases and acute heart failure, and therefore the discovery of potent BRD4 inhibitors with novel scaffolds is highly desirable. In this study, the crystalline water molecules in BRD4 involved in ligand binding were analyzed first, and the simulation results suggest that several conserved crystalline water molecules are quite essential to keep the stability of the crystalline water network and therefore they need to be reserved in structure-based drug design. Then, a docking-based virtual screening workflow with the consideration of the conserved crystalline water network in the binding pocket was utilized to identify the potential inhibitors of BRD4. The in vitro fluorescence resonance energy transfer (HTRF) binding assay illustrates that 4 hits have good inhibitory activity against BRD4 in the micromolar regime, including three compounds with IC50 values below 5 µM and one below 1 µM (0.37 µM). The structural analysis demonstrates that three active compounds possess novel scaffolds. Moreover, the interaction patterns between the hits and BRD4 were characterized by molecular dynamics simulations and binding free energy calculations, and then several suggestions for the further optimization of these hits were proposed.


Assuntos
Simulação de Acoplamento Molecular , Proteínas Nucleares/química , Fatores de Transcrição/química , Água/química , Proteínas de Ciclo Celular , Cristalização , Transferência Ressonante de Energia de Fluorescência , Humanos , Simulação de Dinâmica Molecular , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores
19.
Int Immunopharmacol ; 76: 105921, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31600692

RESUMO

Drug resistance substantially limits the curative capability of chemotherapy in head and neck cancers such as oral squamous cell carcinoma. Immunosuppression is considered a potential cause of drug resistance. A key discovery in the past decade is that chemotherapeutics can alter tumor cell immunogenicity via inducing release of damage-associated molecular patterns (DAMPs), including ecto-calreticulin (ecto-CALR), high mobility group box 1 (HMGB1) and ATP, causing tumor cells to die in a manner known as bona fide immunogenic apoptosis or immunogenic cell death (ICD). Intriguingly, JQ1 was found in this study to exhibit therapeutic potential in tongue squamous cell carcinoma (TSCC) by inducing ICD. JQ1 induced significant release of calreticulin (CALR), HMGB1 and ATP from Cal27 and SCC7 cells in vitro. Immature dendritic cells (Im-DCs) cocultured with JQ1-pretreated Cal27 cells exhibited significant upregulation of mature markers on their surface and an increase in the secretion of cytokines. In vivo experiments demonstrated that JQ1-pretreated dying SCC7 cells protected immunocompetent mice from rechallenge of SCC7 cells. Intravenous injection of JQ1 efficiently reduced tumor growth and increased tumor-infiltration of CD3+/CD8+ T cells in C3H mice.


Assuntos
Antineoplásicos/uso terapêutico , Azepinas/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Neoplasias da Língua/tratamento farmacológico , Fatores de Transcrição/antagonistas & inibidores , Triazóis/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Proteínas de Ciclo Celular/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Proteína HMGB1/metabolismo , Humanos , Camundongos Endogâmicos C3H , Camundongos Nus , Proteínas Nucleares/genética , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Neoplasias da Língua/imunologia , Neoplasias da Língua/patologia , Fatores de Transcrição/genética , Triazóis/farmacologia
20.
Mol Cell ; 76(4): 617-631.e4, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31564557

RESUMO

Spt5 is a conserved and essential transcription elongation factor that promotes promoter-proximal pausing, promoter escape, elongation, and mRNA processing. Spt5 plays specific roles in the transcription of inflammation and stress-induced genes and tri-nucleotide expanded-repeat genes involved in inherited neurological pathologies. Here, we report the identification of Spt5-Pol II small-molecule inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on promoter-proximal pausing, NF-κB activation, and expanded-repeat huntingtin gene transcription. Using SPIs, we identified Spt5 target genes that responded with profoundly diverse kinetics. SPIs uncovered the regulatory role of Spt5 in metabolism via GDF15, a food intake- and body weight-inhibitory hormone. SPIs further unveiled a role for Spt5 in promoting the 3' end processing of histone genes. While several SPIs affect all Spt5 functions, a few inhibit a single one, implying uncoupling and selective targeting of Spt5 activities. SPIs expand the understanding of Spt5-Pol II functions and are potential drugs against metabolic and neurodegenerative diseases.


Assuntos
Núcleo Celular/efeitos dos fármacos , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , RNA Polimerase II/metabolismo , Transcrição Genética/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Fatores de Elongação da Transcrição/antagonistas & inibidores , Regiões 3' não Traduzidas , Animais , Núcleo Celular/enzimologia , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Descoberta de Drogas/métodos , Metabolismo Energético/efeitos dos fármacos , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Histonas/genética , Histonas/metabolismo , Humanos , Proteína Huntingtina/biossíntese , Proteína Huntingtina/genética , Células Jurkat , Células MCF-7 , Camundongos Transgênicos , Mutação , NF-kappa B/biossíntese , NF-kappa B/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Polimerase II/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
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