RESUMO
Gemcitabine is a nucleoside analog widely used as an anticancer agent against several types of cancer. Although gemcitabine sometimes shows excellent effectiveness, cancer cells are often poorly responsive to or resistant to the drug. Recently, specific strains or dysbiosis of the human microbiome were correlated with drug reactivity and resistance acquisition. Therefore, we aimed to identify antibiotic compounds that can modulate the microbiome to enhance the responsiveness to gemcitabine. To achieve this, we confirmed the gemcitabine responsiveness based on public data and conducted drug screening on a set of 250 antibiotics compounds. Subsequently, we performed experiments to investigate whether the selected compounds could enhance the responsiveness to gemcitabine. First, we grouped a total of seven tumor cell lines into resistant and sensitive group based on the IC50 value (1 µM) of gemcitabine obtained from the public data. Second, we performed high-throughput screening with compound treatments, identifying seven compounds from the resistant group and five from the sensitive group based on dose dependency. Finally, the combination of the selected compound, puromycin dihydrochloride, with gemcitabine in gemcitabine-resistant cell lines resulted in extensive cell death and a significant increase in cytotoxic efficacy. Additionally, mRNA levels associated with cell viability and stemness were reduced. Through this study, we screened antibiotics to further improve the efficacy of existing anticancer drugs and overcome resistance. By combining existing anticancer agents and antibiotic substances, we hope to establish various drug combination therapies and ultimately improve cancer treatment efficacy.
Assuntos
Antibacterianos , Desoxicitidina , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Gencitabina , Ensaios de Triagem em Larga Escala , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Humanos , Ensaios de Triagem em Larga Escala/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Antibacterianos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Puromicina/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Sinergismo Farmacológico , Antineoplásicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion in the protein huntingtin (HTT) [55]. While the final pathological consequence of HD is the neuronal cell death in the striatum region of the brain, it is still unclear how mutant HTT (mHTT) causes synaptic dysfunctions at the early stage and during the progression of HD. Here, we discovered that the basal activity of focal adhesion kinase (FAK) is severely reduced in a striatal HD cell line, a mouse model of HD, and the human post-mortem brains of HD patients. In addition, we observed with a FRET-based FAK biosensor [59] that neurotransmitter-induced FAK activation is decreased in HD striatal neurons. Total internal reflection fluorescence (TIRF) imaging revealed that the reduced FAK activity causes the impairment of focal adhesion (FA) dynamics, which further leads to the defect in filopodial dynamics causing the abnormally increased number of immature neurites in HD striatal neurons. Therefore, our results suggest that the decreased FAK and FA dynamics in HD impair the proper formation of neurites, which is crucial for normal synaptic functions [52]. We further investigated the molecular mechanism of FAK inhibition in HD and surprisingly discovered that mHTT strongly associates with phosphatidylinositol 4,5-biphosphate, altering its normal distribution at the plasma membrane, which is crucial for FAK activation [14, 60]. Therefore, our results provide a novel molecular mechanism of FAK inhibition in HD along with its pathological mechanism for synaptic dysfunctions during the progression of HD.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Doença de Huntington , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Adesões Focais/metabolismo , Adesões Focais/patologia , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/patologia , Camundongos , Neuritos/patologia , Neurônios/patologiaRESUMO
ERG-associated protein with the SET domain (ESET/SET domain bifurcated 1/SETDB1/KMT1E) is a histone lysine methyltransferase (HKMT) and it preferentially tri-methylates lysine 9 of histone H3 (H3K9me3). SETDB1/ESET leads to heterochromatin condensation and epigenetic gene silencing. These functional changes are reported to correlate with Huntington's disease (HD) progression and mood-related disorders which make SETDB1/ESET a viable drug target. In this context, the present investigation was performed to identify novel peptide-competitive small molecule inhibitors of the SETDB1/ESET by a combined in silico-in vitro approach. A ligand-based pharmacophore model was built and employed for the virtual screening of ChemDiv and Asinex database. Also, a human SETDB1/ESET homology model was constructed to supplement the data further. Biological evaluation of the selected 21 candidates singled out 5 compounds exhibiting a notable reduction of the H3K9me3 level via inhibitory potential of SETDB1/ESET activity in SETDB1/ESET-inducible cell line and HD striatal cells. Later on, we identified two compounds as final hits that appear to have neuronal effects without cytotoxicity based on the result from MTT assay. These compounds hold the calibre to become the future lead compounds and can provide structural insights into more SETDB1/ESET-focused drug discovery research. Moreover, these SETDB1/ESET inhibitors may be applicable for the preclinical study to ameliorate neurodegenerative disorders via epigenetic regulation.
Assuntos
Derivados de Benzeno/química , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas Metiltransferases/antagonistas & inibidores , Derivados de Benzeno/farmacologia , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular , Simulação por Computador , Bases de Dados de Produtos Farmacêuticos , Humanos , Ligantes , Metilação , Simulação de Acoplamento Molecular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ligação Proteica , Proteínas Metiltransferases/metabolismo , Relação Estrutura-AtividadeRESUMO
Controlling airborne microorganisms has become increasingly important with increase in human indoor activities, epidemic disease outbreaks, and airborne pathogen transmission. Treatments using antimicrobial nanoparticles have shown promise because of the high surface-to-volume ratio of nanoparticles compared to their bulk counterparts, and their unique physical and chemical properties. In this study, hybrid nanostructures of multi-walled carbon nanotubes (MWCNTs) coated with antimicrobial, natural product (NP) nanoparticles were synthesized using a twin-head electrospray system (THES). The coated nanoparticles were then used in antimicrobial air filters to increase their antimicrobial efficiency. Electrosprayed droplets were converted to NP nanoparticles and MWCNTs through ethanol evaporation. Oppositely charged NP nanoparticles and MWCNTs were coagulated via Coulombic collisions to form hybrid nanoparticles that were deposited continuously onto an air filter medium. The size distribution and composition of the hybrid NP/MWCNT particles were characterized using a wide-range particle spectrometer (WPS) and transmission electron microscope (TEM). The concentration of hybrid NP/MWCNT nanoparticles was lower than that of NP nanoparticles but higher than that of MWCNTs and showed a bimodal size distribution with peak diameters of 21.1 and 49 nm. TEM analyses confirmed that the NP nanoparticles were attached to the MWCNT surface with a density of ~4-9 particles/MWCNT. When deposited onto the filter medium, NP/MWCNT particles formed dendrites on the filter׳s fiber surface. The filtration efficiency and pressure drop of the NP/MWCNT-coated filters were higher than those of pristine, NP nanoparticles-coated or MWCNTs-coated filters. The hybrid filter also exhibited stronger antimicrobial activity than those of NP or MWCNT-coated filters at identical deposited volumes (1.1×10-2 cm3/cm2 filter). Ninety-five percent of the tested bacterial aerosols were inactivated on the NP/MWCNTs filter while only <70% were inactivated on NP- or MWCNT-coated filters.
RESUMO
X-linked inhibitor of apoptosis protein (XIAP) deficiency causes refractory inflammatory bowel disease. The XIAP protein plays a pivotal role in the pro-inflammatory response through the nucleotide-binding oligomerization domain-containing signaling pathway that is important in mucosal homeostasis. We analyzed the molecular mechanism of non-synonymous pathogenic variants (PVs) of XIAP BIR2 domain. We generated N-terminally green fluorescent protein-tagged XIAP constructs of representative non-synonymous PVs. Co-immunoprecipitation and fluorescence cross-correlation spectroscopy showed that wild-type XIAP and RIP2 preferentially interacted in live cells, whereas all non-synonymous PV XIAPs failed to interact properly with RIP2. Structural analysis showed that various structural changes by mutations, such as hydrophobic core collapse, Zn-finger loss, and spatial rearrangement, destabilized the two loop structures (174-182 and 205-215) that critically interact with RIP2. Subsequently, it caused a failure of RIP2 ubiquitination and loss of protein deficiency by the auto-ubiquitination of all XIAP mutants. These findings could enhance our understanding of the role of XIAP mutations in XIAP-deficient inflammatory bowel disease and may benefit future therapeutic strategies.
Assuntos
Doenças Inflamatórias Intestinais , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X , Humanos , Proteínas de Fluorescência Verde , Homeostase , Doenças Inflamatórias Intestinais/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
Acute myeloid leukemia (AML) is the most common type of hematological disease in adults, and has a very poor outcome [1]. Based on its wide range of efficacy in AML models, a small-molecule inhibitor of the anti-apoptotic protein BCL-2, venetoclax (ABT-199/GDC-0199), was developed for clinical trials. However, venetoclax showed limited monotherapy activity [2]. The overexpression of myeloid cell leukemia sequence-1 protein (Mcl-1)-due to mutations in Fms-like tyrosine kinase 3 internal tandem duplication (FLT-3 ITD)-was considered to be the main reason for low efficacy of venetoclax in clinical trials [3-5]. To achieve venetoclax sensitization in AML, targeting CDK-9 with venetoclax is a promising therapeutic strategy. In this study, we developed A09-003 as a potent inhibitor of CDK-9, with an IC50 value of 16 nM. A09-003 inhibited cell proliferation in various leukemia cell lines. In particular, the proliferation inhibitory effect of A09-003 was most potent in MV4-11 and Molm-14 cells, harboring the FLT-3 ITD mutation with a high expression profile of Mcl-1. Marker analysis revealed that A09-003 reduced CDK-9 phosphorylation and reduced RNA polymerase II activity with decreased Mcl-1 expression. Finally, combining A09-003 with venetoclax induced apoptotic cell death in a synergistic manner. In summary, this study shows the potential of A09-003 in AML therapy.
Assuntos
Apoptose , Leucemia Mieloide Aguda , Adulto , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Quinases Ciclina-Dependentes , Células Mieloides/metabolismoRESUMO
ANO1, a Ca2+-activated chloride channel, is highly expressed in glioblastoma cells and its surface expression is involved in their migration and invasion. However, the regulation of ANO1 surface expression in glioblastoma cells is largely unknown. In this study, we found that Ca2+/Calmodulin-dependent protein kinase II (CaMKII) ß specifically enhances the surface expression and channel activity of ANO1 in U251 glioblastoma cells. When KN-93, a CaMKII inhibitor, was used to treat U251 cells, the surface expression and channel activity of ANO1 were significantly reduced. Only CaMKIIß, among the four CaMKII isoforms, increased the surface expression and channel activity of ANO1 in a heterologous expression system. Additionally, gene silencing of CaMKIIß suppressed the surface expression and channel activity of ANO1 in U251 cells. Moreover, gene silencing of CaMKIIß or ANO1 prominently reduced the migration and invasion of U251 and U87 MG glioblastoma cells. We thus conclude that CaMKIIß plays a specific role in the surface expression of ANO1 and in the ANO1-mediated tumorigenic properties of glioblastoma cells, such as migration and invasion.
Assuntos
Anoctamina-1/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Glioblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Anoctamina-1/genética , Transporte Biológico , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/genética , Humanos , Proteínas de Neoplasias/genéticaRESUMO
14-3-3γ plays diverse roles in different aspects of cellular processes. Especially in the brain where 14-3-3γ is enriched, it has been reported to be involved in neurological and psychiatric diseases (e.g. Williams-Beuren syndrome and Creutzfeldt-Jakob disease). However, behavioral abnormalities related to 14-3-3γ deficiency are largely unknown. Here, by using 14-3-3γ deficient mice, we found that homozygous knockout mice were prenatally lethal, and heterozygous mice showed developmental delay relative to wild-type littermate mice. In addition, in behavioral analyses, we found that 14-3-3γ heterozygote mice display hyperactive and depressive-like behavior along with more sensitive responses to acute stress than littermate control mice. These results suggest that 14-3-3γ levels may be involved in the developmental manifestation of related neuropsychiatric diseases. In addition, 14-3-3γ heterozygote mice may be a potential model to study the molecular pathophysiology of neuropsychiatric symptoms.
RESUMO
Emerging evidences suggest that phospholipid metabolism is altered in Alzheimer's disease (AD), but molecular mechanisms on how this affects neurodegeneration in AD is poorly understood. SHIP2 is a phosphoinositide-metabolizing enzyme, which dephosphorylates PI(3,4,5)P3 resulting to PI(3,4)P2, and it has been recently shown that Aß directly increases the activity of SHIP2. Here we monitored, utilizing fluorescent SHIP2 biosensor, real-time increase of PI(3,4)P2-containing vesicles in HT22 cells treated with Aß. Interestingly, PI(3,4)P2 is accumulated at late endosomes and lysosomal vesicles. We further discovered that ARAP3 can be attracted to PI(3,4)P2-positive mature endosomes via its PH domain and this facilitates the degradation of ARAP3. The reduced level of ARAP3 then causes RhoA hyperactivation and filamentous actin, which are critical for neurodegeneration in AD. These results provide a novel molecular link between Aß and actin disruption through dysregulated phosphoinositide metabolism, and the SHIP2-PI(3,4)P2-ARAP3-RhoA signaling pathway can be considered as new therapeutic targets for synaptic dysfunctions in Alzheimer's disease.
Assuntos
Citoesqueleto de Actina/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Linhagem Celular , Endossomos/genética , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Fosfatos de Fosfatidilinositol/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Platelet-derived growth factor receptor (PDGFR) senses extracellular growth factors and transfer the signals inside the cells regulating cell proliferation, migration and survival. It has been controversial at which membrane microdomains PDGFRs reside and how they control such diverse intracellular signaling pathways. Here, we developed a novel PDGFR biosensor based on fluorescence resonance energy transfer (FRET), which can detect the real-time PDGFR activity in live cells with high spatiotemporal resolutions. To study subcellular PDGFR activity at membrane microdomains, this PDGFR biosensor was further targeted in or outside lipid rafts via different lipid modification signals. The results suggest that, in response to PDGF stimulation, PDGFR activity is evenly distributed at different membrane microdomains, while integrin-mediated signaling events have inhibitory effects on the activation of PDGFR specifically located in lipid rafts but not outside rafts, implying the role of lipid microdomains as segregated signaling platforms.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Microdomínios da Membrana/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Técnicas Biossensoriais , Células HEK293 , Humanos , Integrinas/metabolismo , Camundongos , Receptores do Fator de Crescimento Derivado de Plaquetas/deficiência , Transdução de SinaisRESUMO
In this study, we demonstrated an antimicrobial nanoparticle-coated electrostatic (ES) air filter. Antimicrobial natural-product Sophora flavescens nanoparticles were produced using an aerosol process, and were continuously deposited onto the surface of air filter media. For the electrostatic activation of the filter medium, a corona discharge electrification system was used before and after antimicrobial treatment of the filter. In the antimicrobial treatment process, the deposition efficiency of S. flavescens nanoparticles on the ES filter was ~12% higher than that on the pristine (Non-ES) filter. In the evaluation of filtration performance using test particles (a nanosized KCl aerosol and submicron-sized Staphylococcus epidermidis bioaerosol), the ES filter showed better filtration efficiency than the Non-ES filter. However, antimicrobial treatment with S. flavescens nanoparticles affected the filtration efficiency of the filter differently depending on the size of the test particles. While the filtration efficiency of the KCl nanoparticles was reduced on the ES filter after the antimicrobial treatment, the filtration efficiency was improved after the recharging process. In summary, we prepared an antimicrobial ES air filter with >99% antimicrobial activity, ~92.5% filtration efficiency (for a 300-nm KCl aerosol), and a ~0.8 mmAq pressure drop (at 13 cm/s). This study provides valuable information for the development of a hybrid air purification system that can serve various functions and be used in an indoor environment.
Assuntos
Filtros de Ar , Anti-Infecciosos , Nanopartículas , Eletricidade Estática , Microbiologia do ArRESUMO
Activated carbon fiber (ACF) filters have a wide range of applications, including air purification, dehumidification, and water purification, due to their large specific surface area, high adsorption capacity and rate, and specific surface reactivity. However, when airborne microorganisms such as bacteria and fungi adhere to the carbon substrate, ACF filters can become a source of microbial contamination, and their filter efficacy declines. Antimicrobial treatments are a promising means of preventing ACF bio-contamination. In this study, we demonstrate the use of Sophora flavescens in antimicrobial nanoparticles coated onto ACF filters. The particles were prepared using an aerosol process consisting of nebulization-thermal drying and particle deposition. The extract from S. flavescens is an effective, natural antimicrobial agent that exhibits antibacterial activity against various pathogens. The efficiency of Staphylococcus epidermidis inactivation increased with the concentration of S. flavescens nanoparticles in the ACF filter coating. The gas adsorption efficiency of the coated antimicrobial ACF filters was also evaluated using toluene. The toluene-removal capacity of the ACF filters remained unchanged while the antimicrobial activity was over 90% for some nanoparticle concentrations. Our results provide a scientific basis for controlling both bioaerosol and gaseous pollutants using antimicrobial ACF filters coated with S. flavescens nanoparticles.