RESUMO
There is accumulating evidence that selective serotonin reuptake inhibitors (SSRIs), clinically used as antidepressants, have a beneficial effect on inflammatory diseases such as coronavirus disease 2019 (COVID-19). We previously compared the inhibitory effects of five U.S. Food and Drug Administration (FDA)-approved SSRIs on the production of an inflammatory cytokine, interleukin-6 (IL-6), and concluded that fluoxetine (FLX) showed the most potent anti-inflammatory activity. Here, we investigated the structure-activity relationship of FLX for anti-inflammatory activity towards J774.1 murine macrophages. FLX suppressed IL-6 production induced by the TLR3 agonist polyinosinic-polycytidylic acid (poly(I : C)) with an IC50 of 4.76 µM. A derivative of FLX containing chlorine instead of the methylamino group lacked activity, suggesting that the methylamino group is important for the anti-inflammatory activity. FLX derivatives bearing an N-propyl or N-(pyridin-3-yl)methyl group in place of the N-methyl group exhibited almost the same activity as FLX. Other derivatives showed weaker activity, and the N-phenyl and N-(4-trifluoromethyl)benzyl derivatives were inactive. The chlorine-containing derivative also lacked inhibitory activity against TLR9- or TLR4-mediated IL-6 production. These derivatives showed similar structure-activity relationships for TLR3- and TLR9-mediated inflammatory responses. However, the activities of all amino group-containing derivatives against the TLR4-mediated inflammatory response were equal to or higher than the activity of FLX. These results indicate that the substituent at the nitrogen atom in FLX strongly influences the anti-inflammatory effect.
Assuntos
Anti-Inflamatórios , Fluoxetina , Interleucina-6 , Relação Estrutura-Atividade , Animais , Fluoxetina/farmacologia , Camundongos , Interleucina-6/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Linhagem Celular , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Citocinas/metabolismo , Receptor 3 Toll-Like/metabolismo , Poli I-C/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/química , Inflamação/tratamento farmacológicoRESUMO
Receptor for advanced glycation end-products (RAGE) and Toll-like receptors (TLRs) are potential therapeutic targets in the treatment of acute and chronic inflammatory diseases. We previously reported that trimebutine, a spasmolytic drug, suppresses RAGE pro-inflammatory signaling pathway in macrophages. The aim of this study was to convert trimebutine to a new small molecule using in silico 3D pharmacophore similarity search, and dissect the mechanistic anti-inflammatory basis. Of note, a unique 3-styrylchromone (3SC), 7-methoxy-3-trimethoxy-SC (7M3TMSC), converted from trimebutine 3D pharmacophore potently suppressed both high mobility group box 1-RAGE and lipopolysaccharide-TLR4 signaling pathways in macrophage-like RAW264.7 cells. More importantly, 7M3TMSC inhibited the phosphorylation of extracellular signaling-regulated kinase 1 and 2 (ERK1/2) and downregulated the production of cytokines, such as interleukin-6. Furthermore, 3D pharmacophore-activity relationship analyses revealed that the hydrogen bond acceptors of the trimethoxy groups in a 3-styryl moiety and the 7-methoxy-group in a chromone moiety in this compound are significant in the dual anti-inflammatory activity. Thus, 7M3TMSC may provide an important scaffold for the development of a new type of anti-inflammatory dual effective drugs targeting RAGE/TLR4-ERK1/2 signaling.
Assuntos
Anti-Inflamatórios/farmacologia , Cromonas/farmacologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptor 4 Toll-Like/metabolismo , Trimebutina/farmacologia , Animais , Anti-Inflamatórios/química , Cromonas/química , Proteína HMGB1/metabolismo , Humanos , Camundongos , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Trimebutina/químicaRESUMO
Because of the critical roles of Toll-like receptors (TLRs) and receptor for advanced glycation end-products (RAGE) in the pathophysiology of various acute and chronic inflammatory diseases, continuous efforts have been made to discover novel therapeutic inhibitors of TLRs and RAGE to treat inflammatory disorders. A recent study by our group has demonstrated that trimebutine, a spasmolytic drug, suppresses the high mobility group box 1âRAGE signaling that is associated with triggering proinflammatory signaling pathways in macrophages. Our present work showed that trimebutine suppresses interleukin-6 (IL-6) production in lipopolysaccharide (LPS, a stimulant of TLR4)-stimulated macrophages of RAGE-knockout mice. In addition, trimebutine suppresses the LPS-induced production of various proinflammatory cytokines and chemokines in mouse macrophage-like RAW264.7 cells. Importantly, trimebutine suppresses IL-6 production induced by TLR2-and TLR7/8/9 stimulants. Furthermore, trimebutine greatly reduces mortality in a mouse model of LPS-induced sepsis. Studies exploring the action mechanism of trimebutine revealed that it inhibits the LPS-induced activation of IL-1 receptor-associated kinase 1 (IRAK1), and the subsequent activations of extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and nuclear factor-κB (NF-κB). These findings suggest that trimebutine exerts anti-inflammatory effects on TLR signaling by downregulating IRAK1âERK1/2âJNK pathway and NF-κB activity, thereby indicating the therapeutic potential of trimebutine in inflammatory diseases. Therefore, trimebutine can be a novel anti-inflammatory drug-repositioning candidate and may provide an important scaffold for designing more effective dual anti-inflammatory drugs that target TLR/RAGE signaling.
Assuntos
Anti-Inflamatórios/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Trimebutina/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Quimiocinas/metabolismo , Feminino , Interleucina-6/metabolismo , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células RAW 264.7 , Receptor para Produtos Finais de Glicação Avançada/deficiência , Receptor para Produtos Finais de Glicação Avançada/genética , Sepse/induzido quimicamente , Sepse/tratamento farmacológico , Trimebutina/uso terapêuticoRESUMO
We previously identified papaverine as an inhibitor of receptor for advanced glycation end-products (RAGE) and showed its suppressive effect on high mobility group box 1 (HMGB1)-mediated responses to inflammation. Here, we found trimebutine to be a 3D pharmacophore mimetics of papaverine. Trimebutine was revealed to have more potent suppressive effects on HMGB1-induced production of pro-inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α in macrophage-like RAW264.7 cells and mouse bone marrow primarily differentiated macrophages than did papaverine. However, the inhibitory effect of trimebutine on the interaction of HMGB1 and RAGE was weaker than that of papaverine. Importantly, mechanism-of-action analyses revealed that trimebutine strongly inhibited the activation of RAGE downstream inflammatory signaling pathways, especially the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), which are mediator/effector kinases recruited to the intracellular domain of RAGE. Consequently, the activation of Jun amino terminal kinase, which is an important effector kinase for the up-regulation of pro-inflammatory cytokines, was inhibited. Taken together, these results suggest that trimebutine may exert its suppressive effect on the HMGB1-RAGE inflammatory signal pathways by strongly blocking the recruitment of ERK1/2 to the intracellular tail domain of RAGE in addition to its weak inhibition of the extracellular interaction of HMGB1 with RAGE. Thus, trimebutine may provide a unique scaffold for the development of novel dual inhibitors of RAGE for inflammatory diseases.
Assuntos
Proteína HMGB1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Trimebutina/farmacologia , Animais , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Janus Quinases/antagonistas & inibidores , Macrófagos , Camundongos , Papaverina/química , Papaverina/farmacologia , Células RAW 264.7 , Trimebutina/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Poly(ADP-ribose) glycohydrolase (PARG) plays an essential role in poly(ADP-ribose) (PAR) turnover, and thereby regulating DNA transactions, such as DNA repair, replication, transcription and recombination. Here, we examined the inhibitory activities of 6-hydroxy-3H-xanthene-3-one (HXO) derivatives and analyzed their binding modes in the active site of PARG by in silico docking study. Among the derivatives, Rose Bengal was found to be the most potent inhibitor of PARG and its halogen groups were revealed to cooperatively potentiate the inhibitory activity. Importantly, the binding mode of Rose Bengal occupied the active site of PARG revealed the presence of unique "Sandwich" residues of Asn869 and Tyr792, which enable the inhibitor to bind tightly with the active pocket. This sandwich interaction could stabilize the π-π interactions of HXO scaffold with Phe902 and Tyr795. In addition, to increase the binding affinity, the iodine and chlorine atoms of this inhibitor could contribute to the inducing of favorable disorders, which promote an entropy boost on the active site of PARG for structural plasticity, and making the stable configuration of HXO scaffold in the active site, respectively, as judged by the analysis of binding free energy. These results provide new insights into the active site of PARG and an additional opportunity for designing selective PARG inhibitors.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Simulação de Acoplamento Molecular , Xantenos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/metabolismo , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Xantenos/químicaRESUMO
Alzheimer's disease (AD) is pathologically characterized by accumulation of amyloid ß (Aß) and hyperphosphorylated tau, and thereby induction of neuronal cell death. The Aß-induced neuronal cell death has been shown to occur by several modes, such as apoptosis, necrosis, and necroptosis. Interestingly, in AD patients, the brain and serum levels of brain-derived neurotrophic factor (BDNF) have been reported to be significantly decreased. However, the relationship between Aß and BDNF in the onset of AD remains to be fully understood. Here, we used neuron-like differentiated human neuroblastoma SH-SY5Y (ndSH-SY5Y) cells to study the neurotoxicity of self-aggregated Aß1-42 peptide under different concentrations of BDNF in the culture medium. Importantly, decreasing levels of BDNF caused a considerable suppression in the extension of neurite length. Furthermore, only under low levels of BDNF, the aggregated Aß was revealed to induce neurite fragmentation and neuronal cell death in ndSH-SY5Y cells. Notably, the aggregated Aß and low levels of BDNF-induced neuronal cell death was characterized at least as caspase-6 dependent cell death and necroptosis. These results indicate that our ndSH-SY5Y cell system, cultured under decreasing levels of BDNF and aggregated Aß, has the potential to be applied in the analysis of the molecular mechanisms of the progressive neurodegenerative processes of AD and the discovery of neuroprotective drug candidates.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Neurônios/metabolismo , Agregação Patológica de Proteínas/metabolismo , Doença de Alzheimer/patologia , Morte Celular , Linhagem Celular Tumoral , Humanos , Modelos Biológicos , Neurônios/patologiaRESUMO
Cancer cells' resistance to drugs remains an important problem affecting cancer treatment strategies. We previously studied the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866's resistance mechanisms in the human colorectal cancer HCT116 cells. We established an acquired FK866-resistant cell line, HCT116RFK866. In this study, we investigated gene mutations in parental HCT116 and HCT116RFK866 cells using exome sequencing technology. The results indicated cluster genes related to NAD+ biosynthesis (including NAMPT), DNA repair, and ATP-binding cassette transporters were differentially altered in these cells. Interestingly, HCT116RFK866 cells, which are resistant to other class NAMPT inhibitors, were more sensitive to the anticancer 5-fluorouracil and cisplatin and γ-ray irradiation compared to parental HCT116 cells. This higher sensitivity appears to cause a genetic change in the identified gene clusters by resistance to the NAMPT inhibitor FK866. Collectively, these novel findings provide a better understanding of anticancer candidate NAMPT inhibitors with regard to resistance mechanisms and cancer chemotherapy strategies.
Assuntos
Acrilamidas/farmacologia , Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Piperidinas/farmacologia , Acrilamidas/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Raios gama , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica , Células HCT116 , Humanos , Piperidinas/administração & dosagem , Sequenciamento do ExomaRESUMO
Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is an attractive therapeutic strategy for targeting cancer metabolism. So far, many potent NAMPT inhibitors have been developed and shown to bind to two unique tunnel-shaped cavities existing adjacent to each active site of a NAMPT homodimer. However, cytotoxicities and resistances to NAMPT inhibitors have become apparent. Therefore, there remains an urgent need to develop effective and safe NAMPT inhibitors. Thus, we designed and synthesized two close structural analogues of NAMPT inhibitors, azaindole-piperidine (3a)- and azaindole-piperazine (3b)-motif compounds, which were modified from the well-known NAMPT inhibitor FK866 (1). Notably, 3a displayed considerably stronger enzyme inhibitory activity and cellular potency than did 3b and 1. The main reason for this phenomenon was revealed to be due to apparent electronic repulsion between the replaced nitrogen atom (N1) of piperazine in 3b and the Nδ atom of His191 in NAMPT by our in silico binding mode analyses. Indeed, 3b had a lower binding affinity score than did 3a and 1, although these inhibitors took similar stable chair conformations in the tunnel region. Taken together, these observations indicate that the electrostatic enthalpy potential rather than entropy effects inside the tunnel cavity has a significant impact on the different binding affinity of 3a from that of 3b in the disparate enzymatic and cellular potencies. Thus, it is better to avoid or minimize interactions with His191 in designing further effective NAMPT inhibitors.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Sítios de Ligação , Domínio Catalítico , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Humanos , Indóis/química , Cinética , Simulação de Acoplamento Molecular , Nicotinamida Fosforribosiltransferase/metabolismo , Piperazina/química , Piperidinas/químicaRESUMO
GGAA motifs in the human TP53 and HELB gene promoters play a part in responding to transresveratrol (Rsv) in HeLa S3 cells. This sequence is also present in the 5'upstream region of the human CDC45 gene, which encodes a component of CMG DNA helicase protein complex. The cells were treated with Rsv (20 µM), then transcripts and the translated protein were analyzed by quantitative RTPCR and western blotting, respectively. The results showed that the CDC45 gene and protein expression levels were induced after the treatment. To examine whether they were due to the activation of transcription, a 5'upstream 556bp of the CDC45 gene was cloned and inserted into a multicloning site of the Luciferase (Luc) expression vector. In the present study, various deletion/point mutationintroduced Luc expression plasmids were constructed and they were used for the transient transfection assay. The results showed that the GGAA motif, which is included in a putative RELB protein recognizing sequence, plays a part in the promoter activity with response to Rsv in HeLa S3 cells.
Assuntos
Proteínas de Ciclo Celular , Humanos , Resveratrol/farmacologia , Regiões Promotoras Genéticas , Sequência de Bases , Transfecção , Células HeLa , Proteínas de Ciclo Celular/genéticaRESUMO
Human retinal organoids have become indispensable tools for retinal disease modeling and drug screening. Despite its versatile applications, the long timeframe for their differentiation and maturation limits the throughput of such research. Here, we successfully shortened this timeframe by accelerating human retinal organoid development using unique pharmacological approaches. Our method comprised three key steps: 1) a modified self-formed ectodermal autonomous multizone (SEAM) method, including dual SMAD inhibition and bone morphogenetic protein 4 treatment, for initial neural retinal induction; 2) the concurrent use of a Sonic hedgehog agonist SAG, activin A, and all-trans retinoic acid for rapid retinal cell specification; and 3) switching to SAG treatment alone for robust retinal maturation and lamination. The generated retinal organoids preserved typical morphological features of mature retinal organoids, including hair-like surface structures and well-organized outer layers. These features were substantiated by the spatial immunostaining patterns of several retinal cell markers, including rhodopsin and L/M opsin expression in the outermost layer, which was accompanied by reduced ectopic cone photoreceptor generation. Importantly, our method required only 90 days for retinal organoid maturation, which is approximately two-thirds the time necessary for other conventional methods. These results indicate that thoroughly optimized pharmacological interventions play a pivotal role in rapid and precise photoreceptor development during human retinal organoid differentiation and maturation. Thus, our present method may expedite human retinal organoid research, eventually contributing to the development of better treatment options for various degenerative retinal diseases.
Assuntos
Ativinas , Diferenciação Celular , Proteínas Hedgehog , Organoides , Retina , Transdução de Sinais , Tretinoína , Humanos , Ativinas/farmacologia , Ativinas/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/citologia , Proteínas Hedgehog/metabolismo , Tretinoína/farmacologia , Retina/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
Transcription is one of the most fundamental nuclear functions and is an enzyme complex-mediated reaction that converts DNA sequences into mRNA. Analyzing DNA sequences of 5'-flanking regions of several human genes that respond to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in HL-60 cells, we have identified that the ets (GGAA) motifs are duplicated, overlapped, or clustered within a 500-bp distance from the most 5'-upstream region of the cDNA. Multiple protein factors including Ets family proteins are known to recognize and bind to the GGAA containing sequences. In addition, it has been reported that the ets motifs play important roles in regulation of various promoters. Here, we propose a molecular mechanism, defined by the presence of duplication and multiplication of the GGAA motifs, that is responsible for the initiation of transcription of several genes and for the recruitment of binding proteins to the transcription start site (TSS) of TATA-less promoters.
Assuntos
Proteínas Proto-Oncogênicas c-ets/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sítio de Iniciação de Transcrição , Sequência de Bases , Sítios de Ligação , Células HL-60 , Humanos , Modelos Genéticos , Regiões Promotoras Genéticas , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
DNA helicases are important in the regulation of DNA transaction and thereby various cellular functions. In this study, we developed a cost-effective multiple DNA transfection assay with DEAE-dextran reagent and analyzed the promoter activities of the human DNA helicases. The 5'-flanking regions of the human DNA helicase-encoding genes were isolated and subcloned into luciferase (Luc) expression plasmids. They were coated onto 96-well plate and used for co-transfection with a renilla-Luc expression vector into various cells, and dual-Luc assays were performed. The profiles of promoter activities were dependent on cell lines used. Among these human DNA helicase genes, XPB, RecQL5, and RTEL promoters were activated during TPA-induced HL-60 cell differentiation. Interestingly, duplicated ets (GGAA) elements are commonly located around the transcription start sites of these genes. The duplicated GGAA motifs are also found in the promoters of DNA replication/repair synthesis factor genes including PARG, ATR, TERC, and Rb1. Mutation analyses suggested that the duplicated GGAA-motifs are necessary for the basal promoter activity in various cells and some of them positively respond to TPA in HL-60 cells. TPA-induced response of 44-bp in the RTEL promoter was attenuated by co-transfection of the PU.1 expression vector. These findings suggest that the duplicated ets motifs regulate DNA-repair associated gene expressions during macrophage-like differentiation of HL-60 cells.
Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Elementos Reguladores de Transcrição/genética , Antineoplásicos/farmacologia , Western Blotting , Carcinógenos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , DEAE-Dextrano , Humanos , Luciferases/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional , Transfecção , Tretinoína/farmacologiaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic of the coronavirus disease in 2019. RNA-dependent RNA polymerase (RdRp) plays an essential role in RNA replication and transcription in SARS-CoV-2. In this study, we focused on the RNA template component of viral RdRp structure and analyzed human microRNAs (miRNAs) targeting specific sequences in this RNA. By examining miRNA databases and using an in vitro RNA-RNA interaction assay, we observed hsa-miR-15b-5p interacting with the RNA component of viral RdRp. Our findings provide evidence that hsa-miR15b-5p may suppresses viral infection and proliferation by targeting the RNA template component of SARS-CoV-2 RdRp.
Assuntos
COVID-19/metabolismo , Regulação Viral da Expressão Gênica , MicroRNAs/fisiologia , RNA Polimerase Dependente de RNA/metabolismo , Biomarcadores/metabolismo , COVID-19/enzimologia , COVID-19/genética , COVID-19/virologia , Bases de Dados Genéticas , Humanos , Técnicas In Vitro , SARS-CoV-2 , Transcrição GênicaRESUMO
transResveratrol (Rsv) is a natural compound contained in red wine and grape skins that has various beneficial effects for organisms such as lengthening of their life span. Rsv induces expression of the human TP53 and HELB genes, which are involved in the regulation of DNA maintenance. In the present study, a luciferase expression vector containing 309 bp of the 5' upstream end of the human MCM4 gene was transfected into HeLa S3 cells. A luciferase assay revealed that Rsv treatment increased the minichromosome maintenance 4 (MCM4) gene promoter activity by GCbox and GGAA (TTCC) motifs. Electro phoretic mobility shift assay revealed that the specific binding factor (complex) contains PU.1 (SPI1). Quantitative reverse transcriptionpolymerase chain reaction analysis indicated that MCM4 gene expression was transiently induced by Rsv. Moreover, western blotting revealed that the SP1/PU.1 ratio markedly increased after Rsv treatment, indicating that a balance or profile of these transcription factors may control Rsvinducible initiation of transcription. These observations indicated that the beneficial effects of Rsv can be attributed to induction of the chromosomal DNA maintenance factor encoding gene expression.
Assuntos
Componente 4 do Complexo de Manutenção de Minicromossomo/genética , Proteínas Proto-Oncogênicas/metabolismo , Resveratrol/farmacologia , Transativadores/metabolismo , Linhagem Celular Tumoral , Células HL-60 , Humanos , Componente 4 do Complexo de Manutenção de Minicromossomo/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
The metabolism of poly(ADP-ribose) plays important roles in the nuclear function of mammalian cells. Previously, we analyzed expression of the poly(ADP-ribose) glycohydrolase (PARG) gene during HL-60 cell differentiation and found that expression was greatly reduced by 4 h after 12-O-tetradecanoyl-phorbol-13-acetate (TPA) treatment and returned to the initial level within 20 h. In the present study, a 2.1-kb fragment of the 5'-flanking (promoter) region of the human PARG gene was isolated from the HL-60 genome by polymerase chain reaction and ligated into a luciferase-expression vector, pGL3, to generate the pPARG-Luc#2 reporter plasmid. Deletion analysis revealed that a 75-nt sequence is required for basal promoter activity and TPA responsiveness. Mutations in this 75-nt sequence reduced promoter activity and the TPA response of HL-60 cells. TFSEARCH analysis revealed that Ets family binding motifs are located in the 75-nt sequence. Chromatin immunoprecipitation assay, electrophoretic mobility shift assay and competition analysis indicated that PU.1 (Spi-1) binds to the 75-nt sequence. Moreover, co-transfection of HL-60 cells with a PU.1 expression plasmid and pPARG-Luc indicated that PU.1 down-regulate the PARG promoter. These results suggest that PARG gene expression is modulated by PU.1 during TPA-induced differentiation of HL-60 cells.
Assuntos
Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Glicosídeo Hidrolases/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Região 5'-Flanqueadora , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Deleção de Genes , Glicosídeo Hidrolases/química , Células HL-60 , Humanos , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Alinhamento de Sequência , Acetato de Tetradecanoilforbol/farmacologia , Transativadores/genéticaRESUMO
Poly(ADP-ribosyl)ation is a unique post-translational modification of proteins. The metabolism of poly(ADP-ribose) (PAR) is tightly regulated mainly by poly(ADP-ribose) polymerases (PARP) and poly(ADP-ribose) glycohydrolase (PARG). Accumulating evidence has suggested the biological functions of PAR metabolism in control of many cellular processes, such as cell proliferation, differentiation and death by remodeling chromatin structure and regulation of DNA transaction, including DNA repair, replication, recombination and transcription. However, the physiological roles of the catabolism of PAR catalyzed by PARG remain less understood than those of PAR synthesis by PARP. Noteworthy biochemical studies have revealed the importance of PAR catabolic pathway generating nuclear ATP via the coordinated actions of PARG and ADP-ribose pyrophosphorylase (ADPRPPL) for the driving of DNA repair and the maintenance of DNA replication apparatus while repairing DNA damage. Furthermore, genetic studies have shown the value of PARG as a therapeutic molecular target for PAR-mediated diseases, such as cancer, inflammation and many pathological conditions. In this review, we present the current knowledge of de-poly(ADP-ribosyl)ation catalyzed by PARG focusing on its role in DNA repair, replication and apoptosis. Furthermore, the induction of apoptosis code of DNA replication catastrophe by synthetic lethality of PARG inhibition and the recent progresses regarding the development of small molecule PARG inhibitors and their therapeutic potentials in cancer chemotherapy are highlighted in this review.
Assuntos
Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Inibidores de Glicosídeo Hidrolases/administração & dosagem , Glicosídeo Hidrolases/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Animais , Apoptose/fisiologia , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/fisiologia , Sistemas de Liberação de Medicamentos/tendências , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Neoplasias/enzimologia , Neoplasias/genéticaRESUMO
BACKGROUND/AIM: Nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in the NAD+ biosynthetic pathway, is a drug target of potent anticancer candidates, including FK866 and other reported NAMPT inhibitors. However, it is known that NAMPT point-mutations render resistance to specific NAMPT inhibitors in several cancer cells. We investigated the resistance mechanisms of NAMPT inhibitor FK866 in human colorectal cancer (CRC) cells. MATERIALS AND METHODS: We used CRC human cell line HCT116 to determine the expression profiles of FK866-sensitive parental HCT116 cells and FK866-resistant HCT116 (HCT116RFK866) cells by DNA microarray analysis. The levels of multidrug resistance protein 1 (MDR1) were assessed via western blot. In addition, we analyzed the sensitivity of FK866 in parental HCT116 cells and HCT116RFK866 cells by co-treatment with MDR1 inhibitor verapamil. RESULTS: Our results revealed an association between ATP-binding cassette (ABC) transporter gene ABCB1 and resistance to NAMPT inhibitor FK866 in both HCT116RFK866 cells and parental HCT116 cells. The expression of ABCB1, which encodes MDR1, was lower in HCT116RFK866 cells than in parental HCT116 cells. Furthermore, the protein level of MDR1/ATP-binding cassette sub-family B member 1 (ABCB1) was 0.5-fold lower in HCT116RFK866 cells than in parental HCT116 cells. Additionally, HCT116RFK866 cells showed improved sensitivity to FK866 when co-treated with verapamil, an ABCB1 inhibitor. Interestingly, the efficacy of FK866 in parental HCT116 cells was the same for the treatment with FK866 alone or in combination with verapamil. CONCLUSION: The change in expression of ABCB1 plays a key role in CRC drug resistance to NAMPT inhibitor FK866. This suggests that the MDR1/ABCB1 mechanism may regulate the resistance of anticancer NAMPT inhibitor FK866.
Assuntos
Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Acrilamidas/farmacologia , Neoplasias Colorretais/metabolismo , Citocinas/antagonistas & inibidores , Regulação para Baixo , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Piperidinas/farmacologia , Verapamil/farmacologiaRESUMO
MicroRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at the post-transcriptional level. Several miRNAs are exclusively expressed in skeletal muscle and participate in the regulation of muscle differentiation by interacting with myogenic factors. These miRNAs can be found at high levels in the serum of patients and animal models for Duchenne muscular dystrophy, which is expected to be useful as biomarkers for their clinical conditions. By miRNA microarray analysis, we identified miR-188 as a novel miRNA that is elevated in the serum of the muscular dystrophy dog model, CXMDJ. miR-188 was not muscle-specific miRNA, but its expression was up-regulated in skeletal muscles associated with muscle regeneration induced by cardiotoxin-injection in normal dogs and mice. Manipulation of miR-188 expression using antisense oligo and mimic oligo RNAs alters the mRNA expression of the myogenic regulatory factors, MRF4 and MEF2C. Our results suggest that miR-188 is a new player that participates in the gene regulation process of muscle differentiation and that it may serve as a serum biomarker reflecting skeletal muscle regeneration.
Assuntos
Biomarcadores/sangue , Regulação da Expressão Gênica , MicroRNAs/genética , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Cães , Camundongos , Músculo Esquelético/patologia , Distrofia Muscular Animal/sangue , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/patologia , Mioblastos/citologia , Mioblastos/metabolismoRESUMO
Human promyelocytic HL60 cells can be differentiated into macrophagelike cells by treatment with 12Otetra decanoylphorbol13acetate (TPA). Certain 5' upstream regions of the zinc finger protein (ZNF)encoding genes contain duplicated GGAA motifs, which are frequently found in the TPAresponding gene promoter regions. To examine transcriptional responses to TPA, 5'flanking regions of human zinc finger CCCHtype containing, antiviral, ZNF252, ZNF343, ZNF555, ZNF782 and zinc finger nfx1type containing 1 (ZNFX1) genes were isolated by polymerase chain reaction (PCR) and ligated into a multiplecloning site of the pGL4.10[luc2] vector. Transient transfection and a luciferase assay revealed that the ZNFX1 promoter most prominently responded to the TPA treatment. Deletion and point mutation experiments indicated that the duplicated GGAA motif in the 100bp region positively responded to TPA. In addition, reverse transcriptionquantitative PCR and western blotting showed that the mRNA and protein of ZNFX1 accumulate during the differentiation of HL60 cells. These results indicated that expression of the TPAinducible ZNFX1 gene, which belongs to the group of interferonresponsive genes, is regulated by the cisaction of the duplicated GGAA motif.