RESUMO
Polycomb repressive complex 2 (PRC2) consists of three core subunits, EZH2, EED and SUZ12, and plays pivotal roles in transcriptional regulation. The catalytic subunit EZH2 methylates histone H3 lysine 27 (H3K27), and its activity is further enhanced by the binding of EED to trimethylated H3K27 (H3K27me3). Small-molecule inhibitors that compete with the cofactor S-adenosylmethionine (SAM) have been reported. Here we report the discovery of EED226, a potent and selective PRC2 inhibitor that directly binds to the H3K27me3 binding pocket of EED. EED226 induces a conformational change upon binding EED, leading to loss of PRC2 activity. EED226 shows similar activity to SAM-competitive inhibitors in blocking H3K27 methylation of PRC2 target genes and inducing regression of human lymphoma xenograft tumors. Interestingly, EED226 also effectively inhibits PRC2 containing a mutant EZH2 protein resistant to SAM-competitive inhibitors. Together, we show that EED226 inhibits PRC2 activity via an allosteric mechanism and offers an opportunity for treatment of PRC2-dependent cancers.
Assuntos
Antineoplásicos/farmacologia , Histonas/metabolismo , Lisina/metabolismo , Complexo Repressor Polycomb 2/antagonistas & inibidores , Sulfonas/química , Sulfonas/farmacologia , Triazóis/química , Triazóis/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Antineoplásicos/química , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Histonas/química , Humanos , Lisina/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Relação Estrutura-Atividade , Sulfonas/metabolismo , Triazóis/metabolismo , Células Tumorais CultivadasRESUMO
The Polycomb repressive complex 2 (PRC2) confers transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3). Here, we examined how PRC2 is modulated by histone modifications associated with transcriptionally active chromatin. We provide the molecular basis of histone H3 N terminus recognition by the PRC2 Nurf55-Su(z)12 submodule. Binding of H3 is lost if lysine 4 in H3 is trimethylated. We find that H3K4me3 inhibits PRC2 activity in an allosteric fashion assisted by the Su(z)12 C terminus. In addition to H3K4me3, PRC2 is inhibited by H3K36me2/3 (i.e., both H3K36me2 and H3K36me3). Direct PRC2 inhibition by H3K4me3 and H3K36me2/3 active marks is conserved in humans, mouse, and fly, rendering transcriptionally active chromatin refractory to PRC2 H3K27 trimethylation. While inhibition is present in plant PRC2, it can be modulated through exchange of the Su(z)12 subunit. Inhibition by active chromatin marks, coupled to stimulation by transcriptionally repressive H3K27me3, enables PRC2 to autonomously template repressive H3K27me3 without overwriting active chromatin domains.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Cromatina/genética , Cristalografia por Raios X , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Humanos , Lisina/química , Metilação , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteína 4 de Ligação ao Retinoblastoma/química , Proteína 4 de Ligação ao Retinoblastoma/genética , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Transcrição GênicaRESUMO
Ezh2 (Enhancer of zeste homolog 2) protein is the enzymatic component of the Polycomb repressive complex 2 (PRC2), which represses gene expression by methylating lysine 27 of histone H3 (H3K27) and regulates cell proliferation and differentiation during embryonic development. Recently, hot-spot mutations of Ezh2 were identified in diffused large B-cell lymphomas and follicular lymphomas. To investigate if tumor growth is dependent on the enzymatic activity of Ezh2, we developed a potent and selective small molecule inhibitor, EI1, which inhibits the enzymatic activity of Ezh2 through direct binding to the enzyme and competing with the methyl group donor S-Adenosyl methionine. EI1-treated cells exhibit genome-wide loss of H3K27 methylation and activation of PRC2 target genes. Furthermore, inhibition of Ezh2 by EI1 in diffused large B-cell lymphomas cells carrying the Y641 mutations results in decreased proliferation, cell cycle arrest, and apoptosis. These results provide strong validation of Ezh2 as a potential therapeutic target for the treatment of cancer.
Assuntos
Linfoma Difuso de Grandes Células B/patologia , Complexo Repressor Polycomb 2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Embrião de Mamíferos/citologia , Proteína Potenciadora do Homólogo 2 de Zeste , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Linfoma Difuso de Grandes Células B/genética , Metilação/efeitos dos fármacos , Camundongos , Mutação/genética , Fenótipo , Complexo Repressor Polycomb 2/metabolismo , Bibliotecas de Moléculas Pequenas/química , Ensaio Tumoral de Célula-Tronco , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs that regulate human gene expression at the post-transcriptional level. Growing evidence indicates that the expression profile of miRNAs is highly correlated with the occurrence of human diseases including cancers. Playing important roles in complex gene regulation processes, the aberrant expression pattern of various miRNAs is implicated in different types and even stages of cancer. Besides localizing in cells, many of these miRNAs are found circulating around the body in a wide variety of fluids such as urine, serum and saliva. Surprisingly, these extracellular circulating miRNAs are highly stable and resistant to degradation, and therefore, are considered as promising biomarkers for early cancer diagnostic via noninvasive extraction from body fluids. Unfortunately, the abundance of these small RNAs is ultralow in the body fluids, making it challenging to quantify them in complex sample matrixes. Establishing a sensitive, specific yet simple assay for an accurate quantification of circulating miRNAs is therefore desirable. Our group previously reported a sensitive and specific detection assay of miRNAs in single molecule level with the aid of total internal reflection fluorescence microscopy. In this work, we advanced the assay to differentiate the expression of a nasopharyngeal carcinoma (NPC) up-regulator hsa-mir-205 (mir-205) in serum collected from patients of different stages of NPC. To overcome the background matrix interference in serum, a locked nucleic acid-modified molecular beacon (LNA/MB) was applied as the detection probe to hybridize, capture and detect target mir-205 in serum matrix with enhanced sensitivity and specificity. A detection limit of 500 fM was achieved. The as-developed method was capable of differentiating NPC stages by the level of mir-205 quantified in serum with only 10 µL of serum and the whole assay can be completed in 1 h. The experimental results agreed well with those previously reported whereas the quantity of miR-205 determined by our assay was found comparable to that of quantitative reverse transcription polymerase chain reaction (qRT-PCR), supporting that this assay can be served as a promising noninvasive detection tool for early NPC diagnosis, monitoring and staging.
Assuntos
Biomarcadores Tumorais/sangue , Regulação Neoplásica da Expressão Gênica , MicroRNAs/sangue , Microscopia de Fluorescência/métodos , Neoplasias Nasofaríngeas/diagnóstico , Biomarcadores Tumorais/genética , Carcinoma , Corantes Fluorescentes/química , Humanos , Limite de Detecção , MicroRNAs/genética , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/sangue , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Estadiamento de Neoplasias , Hibridização de Ácido Nucleico , Oligonucleotídeos/químicaRESUMO
We reported a sensitive detection system for measuring DNA-protein interaction at single plasmonic metal nanoparticles level by Localized Scattering Plasmon Resonance (LSPR) spectroscopy. As a proof of concept, DNA molecules were conjugated to gold nanoparticles (AuNPs) through gold-thiol chemistry and the resulted complex was served as single-particle probes of human topoisomerase I (TOPO). By recording the changes in Rayleigh light scattering signal of the individual nanoparticles upon protein binding, DNA-protein interaction was monitored and measured. The λmax shifts in LSPR spectrum of individual AuNP was found to be highly correlated with the amount of TOPO that bound onto. This technique provides a sensitive and high-throughput platform to screen and monitor accurately the specific biomolecular interactions. It is capable of revealing information such as particle-particle variations that might be buried in conventional bulk measurement.
Assuntos
DNA Topoisomerases Tipo I/química , DNA de Cadeia Simples/química , Ácidos Nucleicos Imobilizados/química , Nanopartículas Metálicas/química , Ressonância de Plasmônio de Superfície , Ouro/química , Humanos , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Nanocompostos/química , Tamanho da Partícula , Ligação ProteicaRESUMO
A one-dimensional nanofibrillar array formed by the co-assembly of native and biotin-functionalized beta-amyloid (Aß) peptide was developed for biomolecule sensing. With the presence of biotin moiety, a variety of biomolecular probes can be conjugated onto the nanofibrils, thus converting the protein assembly into a miniature biosensor. In this work, DNA probes were immobilized onto the fibril for the detection of cDNA sequences. The as-developed "DNA-nanoarray" achieved a detection limit at subattomole level (183 fM in 10 µL). This highly sensitive, yet simple, assay requires a trace amount of sample consumption (<10 µL) and is pretreatment-free. In addition, we reported the preparation of alternate-segmented amyloid nanofibrils with multifunctionality. The fibrils hereby serve as an encoded template that can be visualized with various fluorescence labeling dyes for barcode recognition purpose, and, hence, multiplex detection of biomolecules was achieved. Regarding that each protein nanofibril represents a single detection platform, a large number of single fibrils simultaneously are monitored with the dual-color TIRFM in a high-throughput manner.
Assuntos
Técnicas Biossensoriais , DNA/análise , Nanofibras/química , Peptídeos beta-Amiloides/química , Biotina/química , Sondas de DNA/química , Corantes Fluorescentes/química , Proteínas Imobilizadas/química , Fragmentos de Peptídeos/química , Espectrometria de FluorescênciaRESUMO
MicroRNAs (miRNAs) express differently in normal and cancerous tissues and thus are regarded as potent cancer biomarkers for early diagnosis. However, the short length and low abundance of miRNAs have brought challenges to the established detection assay in terms of sensitivity and selectivity. In this work, we present a novel miRNA detection assay in single-molecule level with total internal reflection fluorescence microscopy (TIRFM). It is a solution-based hybridization detection system that does not require pretreatment steps such as sample enrichment or signal amplification. The hsa-miR-21 (miR-21) is chosen as target miRNA for its significant elevated content in a variety of cancers as reported previously. Herein, probes of complementary single-stranded oligonucleotide were hybridized in solution to miR-21 and labeled with fluorescent dye YOYO-1. The fluorescent hybrids were imaged by an electron-multiplying charge-coupled device (EMCCD) coupled TIRFM system and quantified by single-molecule counting. This single molecule detection (SMD) assay shows a good correlation between the number of molecules detected and the factual concentration of miRNA. The detection assay is applied to quantify the miR-21 in extracted total RNA samples of cancerous MCF-7 cells, HepG2 cells, and normal HUVEC cells, respectively. The results agreed very well with those from the prevalent real-time polymerase chain reaction (qRT-PCR) analysis. This assay is of high potential for applications in miRNA expression profiling and early cancer diagnosis.
Assuntos
MicroRNAs/análise , Microscopia de Fluorescência/métodos , Hibridização de Ácido Nucleico/métodos , Benzoxazóis/química , Linhagem Celular , Detecção Precoce de Câncer , Corantes Fluorescentes/química , Células Hep G2 , Humanos , Oligonucleotídeos/química , Compostos de Quinolínio/químicaRESUMO
SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein-ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Antineoplásicos/uso terapêutico , Cães , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Macaca fascicularis , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ratos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Protein tyrosine phosphatase SHP2 is an oncoprotein associated with cancer as well as a potential immune modulator because of its role in the programmed cell death PD-L1/PD-1 pathway. In the preceding manuscript, we described the optimization of a fused, bicyclic screening hit for potency, selectivity, and physicochemical properties in order to further expand the chemical diversity of allosteric SHP2 inhibitors. In this manuscript, we describe the further expansion of our approach, morphing the fused, bicyclic system into a novel monocyclic pyrimidinone scaffold through our understanding of SAR and use of structure-based design. These studies led to the identification of SHP394 (1), an orally efficacious inhibitor of SHP2, with high lipophilic efficiency, improved potency, and enhanced pharmacokinetic properties. We also report other pyrimidinone analogues with favorable pharmacokinetic and potency profiles. Overall, this work improves upon our previously described allosteric inhibitors and exemplifies and extends the range of permissible chemical templates that inhibit SHP2 via the allosteric mechanism.
Assuntos
Aminopiridinas/uso terapêutico , Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Pirimidinonas/uso terapêutico , Administração Oral , Regulação Alostérica , Sítio Alostérico , Aminopiridinas/síntese química , Aminopiridinas/farmacocinética , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Pirimidinonas/síntese química , Pirimidinonas/farmacocinética , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
SHP2 is a nonreceptor protein tyrosine phosphatase within the mitogen-activated protein kinase (MAPK) pathway controlling cell growth, differentiation, and oncogenic transformation. SHP2 also participates in the programed cell death pathway (PD-1/PD-L1) governing immune surveillance. Small-molecule inhibition of SHP2 has been widely investigated, including in our previous reports describing SHP099 (2), which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies, reported here in the first of two papers, led to the identification of multiple 5,6-fused bicyclic scaffolds that bind to the same allosteric tunnel as 2. We demonstrate the structural diversity permitted by the tunnel pharmacophore and culminated in the identification of pyrazolopyrimidinones (e.g., SHP389, 1) that modulate MAPK signaling in vivo. These studies also served as the basis for further scaffold morphing and optimization, detailed in the following manuscript.
Assuntos
Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 2 Anéis/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Regulação Alostérica , Sítio Alostérico , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Pirazóis/síntese química , Pirazóis/metabolismo , Pirimidinonas/síntese química , Pirimidinonas/metabolismo , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
E2F proteins can either activate or repress transcription. Following mitogenic stimulation, repressive E2F4-p130-histone deacetylase complexes dissociate from, while activating species (E2F1, -2, and -3) associate with, target promoters. Histones H3 and H4 simultaneously become hyperacetylated, but it remains unclear whether this is a prerequisite or a consequence of E2F binding. Here, we show that activating E2F species are required for hyperacetylation of target chromatin in human cells. Overexpression of a dominant-negative (DN) E2F1 mutant in serum-stimulated T98G cells blocked all E2F binding, H4 acetylation, and, albeit partially, H3 acetylation. Target gene activation and S-phase entry were also blocked by DN E2F1. Conversely, ectopic activation of E2F1 rapidly induced H3 and H4 acetylation, demonstrating a direct role for E2F in these events. E2F1 was previously shown to bind the histone acetyltransferases (HATs) p300/CBP and PCAF/GCN5. In our hands, ectopically expressed E2F1 also bound the unrelated HAT Tip60 and induced recruitment of five subunits of the Tip60 complex (Tip60, TRRAP, p400, Tip48, and Tip49) to target promoters in vivo. Moreover, E2F-dependent recruitment of Tip60 to chromatin occurred in late G(1) following serum stimulation. We speculate that the activities of multiple HAT complexes account for E2F-dependent acetylation, transcription, and S-phase entry.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Linhagem Celular , Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F4 , Fase G1 , Regulação da Expressão Gênica , Histona Acetiltransferases , Humanos , Cinética , Lisina Acetiltransferase 5 , Mutação , Ligação Proteica , Fase S , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Assessing the functional significance of novel putative oncogenes remains a significant challenge given the limitations of current loss-of-function tools. Here, we describe a method that employs TALEN or CRISPR/Cas9-mediated knock-in of inducible degron tags (Degron-KI) that provides a versatile approach for the functional characterization of novel cancer genes and addresses many of the shortcomings of current tools. The Degron-KI system allows for highly specific, inducible, and allele-targeted inhibition of endogenous protein function, and the ability to titrate protein depletion with this system is able to better mimic pharmacologic inhibition compared with RNAi or genetic knockout approaches. The Degron-KI system was able to faithfully recapitulate the effects of pharmacologic EZH2 and PI3Kα inhibitors in cancer cell lines. The application of this system to the study of a poorly understood putative oncogene, SF3B1, provided the first causal link between SF3B1 hotspot mutations and splicing alterations. Surprisingly, we found that SF3B1-mutant cells are not dependent upon the mutated allele for in vitro growth, but instead depend upon the function of the remaining wild-type alleles. Collectively, these results demonstrate the broad utility of the Degron-KI system for the functional characterization of cancer genes.
Assuntos
Genes Neoplásicos , Neoplasias/genética , Proliferação de Células , Células HCT116 , Humanos , Mutação , Fosfoproteínas/genética , Estabilidade Proteica , Proteólise , Fatores de Processamento de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genéticaRESUMO
MicroRNA (miRNA) has recently emerged as a new and important class of cellular regulators. Strong evidences showed that aberrant expression of miRNA is associated with a broad spectrum of human diseases, such as cancer, diabetes, cardiovascular and psychological disorders. However, the short length and low abundance of miRNA place great challenges for conventional techniques in the miRNA quantification and expression profiling. Here, we report a direct, specific and highly sensitive yet simple detection assay for miRNA without sample amplification. A self-assembled protein nanofibril acted as an online pre-concentrating sensor to detect the target miRNA. Locked nucleic acid (LNA) of complimentary sequence was served as the probe to capture the target miRNA analyte. The quantification was achieved by the fluorescence intensity measured with total internal reflection fluorescence microscopy. A detection limit of 1 pM was achieved with trace amount of sample consumption. This assay showed efficient single-base mismatch discrimination. The applicability of quantifying circulating mir-196a in both normal and cancer patient's serums was also demonstrated.
Assuntos
MicroRNAs/sangue , Microscopia de Fluorescência/métodos , Humanos , Nanoestruturas/química , Sensibilidade e EspecificidadeRESUMO
This is the first work that revealed the neuro-protective effect of functionalized quantum dots against the cytotoxicity induced by beta-amyloid peptides. This study gives insight into the future treatment of Alzheimer's disease. It opens many avenues for the development of the next generation nanotechnology for biomedical and therapeutic applications.
RESUMO
In this paper, we investigated the functional imaging and targeted therapeutic properties of core@multi-shell nanoparticles composed of a superparamagnetic iron oxide (SPIO) core and gold nanorods (GNRs) in the mesoporous silica shells functionalized with folic acid (Fe3O4@SiO2@GNRs@mSiO2-FA). The as-synthesized five-component hybrid nanocomposite was revealed to have insignificant cytotoxicity. Intracellular uptake of the nanoparticles was studied in the folate receptor over-expressing human epidermoid carcinoma of the nasopharynx (KB) cells. Due to their magnetic/optical properties as well as the folate targeting potential, compared with Fe3O4@SiO2@GNRs@mSiO2 nanoparticles, higher cellular uptake efficiency was observed for Fe3O4@SiO2@GNRs@mSiO2-FA nanoparticles in KB cells. Characterizations were achieved using both dark field and magnetic resonance (MR) imaging techniques. The hyperthermia induced by Fe3O4@SiO2@GNRs@mSiO2-FA nanoparticles resulted in a higher cytotoxicity in KB cells. Thus, the Fe3O4@SiO2@GNRs@mSiO2-FA hybrid nanomaterial is an effective and promising MR imaging and photothermal therapy agent for folate-receptor over-expressing cancer cells.
RESUMO
Histone lysine methyltransferase NSD2 (WHSC1/MMSET) is overexpressed frequently in multiple myeloma due to the t(4;14) translocation associated with 15% to 20% of cases of this disease. NSD2 has been found to be involved in myelomagenesis, suggesting it may offer a novel therapeutic target. Here we show that NSD2 methyltransferase activity is crucial for clonogenicity, adherence, and proliferation of multiple myeloma cells on bone marrow stroma in vitro and that NSD2 is required for tumorigenesis of t(4;14)+ but not t(4;14)- multiple myeloma cells in vivo. The PHD domains in NSD2 were important for its cellular activity and biological function through recruiting NSD2 to its oncogenic target genes and driving their transcriptional activation. By strengthening its disease linkage and deepening insights into its mechanism of action, this study provides a strategy to therapeutically target NSD2 in multiple myeloma patients with a t(4;14) translocation.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/genética , Proteínas Repressoras/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Histona-Lisina N-Metiltransferase/genética , Humanos , Masculino , Camundongos , Camundongos SCID , Mieloma Múltiplo/patologia , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Ativação Transcricional , Translocação GenéticaRESUMO
The influence of nanoparticles of various sizes and surface functionalities on the self-assembling fibrillogenesis of beta-amyloid (1-40) peptide was investigated. Functionalized nanoparticles including quantum dots and gold nanoparticles were co-incubated with monomeric Aß(1-40) peptides under seed-mediated growth method to study their influences on the elongation phase of the fibrillogenesis. It is observed that charge-to-surface area ratio of the nanoparticles and the functional moiety and electrostatic charges of the conjugated ligands on the particle surfaces took crucial regulatory role in the Aß(1-40) fibrillogenesis.
Assuntos
Peptídeos beta-Amiloides/química , Nanopartículas Metálicas/química , Fragmentos de Peptídeos/química , Pontos Quânticos , Peptídeos beta-Amiloides/ultraestrutura , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/ultraestruturaRESUMO
The adenovirus E1A oncoprotein promotes proliferation and transformation by binding cellular proteins, including members of the retinoblastoma protein family, the p300/CREB-binding protein transcriptional coactivators, and the p400-TRRAP chromatin-remodeling complex. E1A also promotes apoptosis, in part, by engaging the ARF-p53 tumor suppressor pathway. We show that E1A induces ARF and p53 and promotes apoptosis in normal fibroblasts by physically associating with the retinoblastoma protein and a p400-TRRAP complex and that its interaction with p300 is largely dispensable for these effects. We further show that E1A increases p400 expression and, conversely, that suppression of p400 using stable RNA interference reduces the levels of ARF, p53, and apoptosis in E1A-expressing cells. Therefore, whereas E1A inactivates the retinoblastoma protein, it requires p400 to efficiently promote cell death. These results identify p400 as a regulator of the ARF-p53 pathway and a component of the cellular machinery that couples proliferation to cell death.