RESUMO
This chapter introduces a simple and robust in vitro viability assay to screen bioactive small molecules (e.g., natural, synthetic) against the monomorphic and infective (bloodstream) form of Trypanosoma brucei brucei. The assay relies on a bioluminescent transgenic parasite harboring a genetically encoded copy of a thermostable redshifted firefly luciferase from Photinus pyralis.The major advantages of the assay are simplicity and cost efficiency, along with excellent quality parameters. The bioassay allows estimating parasite numbers and viability (and metabolic state) as a function of bioluminescence (BL) signal. Parasites are grown in the presence of the molecules of interest in a 96-well microplate, and 24 h later, BL is determined with a simple protocol lacking washing steps, using cost-efficient reagents with a reasonable readout time for high-throughput applications.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Medições Luminescentes , Trypanosoma brucei brucei , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Luciferases de Vaga-Lume , Medições Luminescentes/métodos , Trypanosoma brucei brucei/efeitos dos fármacosRESUMO
The main protease, Mpro, of SARS-CoV-2 is required to cleave the viral polyprotein into precise functional units for virus replication and pathogenesis. Here, we report quantitative reporters for Mpro function in living cells in which protease inhibition by genetic or chemical methods results in robust signal readouts by fluorescence (enhanced green fluorescent protein [eGFP]) or bioluminescence (firefly luciferase). These gain-of-signal systems are scalable to high-throughput platforms for quantitative discrimination between Mpro mutants and/or inhibitor potencies as evidenced by validation of several reported inhibitors. Additional utility is shown by single Mpro amino acid variants and structural information combining to demonstrate that both inhibitor conformational dynamics and amino acid differences are able to influence inhibitor potency. We further show that a recent variant of concern (Omicron) has an unchanged response to a clinically approved drug, nirmatrelvir, whereas proteases from divergent coronavirus species show differential susceptibility. Together, we demonstrate that these gain-of-signal systems serve as robust, facile, and scalable assays for live cell quantification of Mpro inhibition, which will help expedite the development of next-generation antivirals and enable the rapid testing of emerging variants. IMPORTANCE The main protease, Mpro, of SARS-CoV-2 is an essential viral protein required for the earliest steps of infection. It is therefore an attractive target for antiviral drug development. Here, we report the development and implementation of two complementary cell-based systems for quantification of Mpro inhibition by genetic or chemical approaches. The first is fluorescence based (eGFP), and the second is luminescence based (firefly luciferase). Importantly, both systems rely upon gain-of-signal readouts such that stronger inhibitors yield higher fluorescent or luminescent signal. The high versatility and utility of these systems are demonstrated by characterizing Mpro mutants and natural variants, including Omicron, as well as a panel of existing inhibitors. These systems rapidly, safely, and sensitively identify Mpro variants with altered susceptibilities to inhibition, triage-nonspecific, or off-target molecules and validate bona fide inhibitors, with the most potent thus far being the first-in-class drug nirmatrelvir.
Assuntos
Antivirais , Proteases 3C de Coronavírus , Inibidores de Proteases , SARS-CoV-2 , Aminoácidos , Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Luciferases de Vaga-Lume , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genéticaRESUMO
Among numerous bioluminescent organisms, firefly is the most studied one. Recent experiment proposed that sulfoluciferin (SLH2 ) may serve as a storage form of luciferin (LH2 ). In the present article, we employed density functional theory calculation to uncover the mechanism and detailed process of the storage and release reactions. Due to lack of available crystallographic structure of the related enzyme, the calculation was performed on a model system. For the storage reaction, possible amino acid residues were used for imitating the protein environment. For the release reaction, the dielectric constant of 3.0 was employed to simulate the polarity of the protein cavity. The computational results indicated that the reactions from LH2 to SLH2 and from SLH2 to LH2 are both exergonic, which favor the storage and release processes and coincide with the experimental observation. Basing on experimental and current theoretical study, we supplemented the stages of LH2 storage and release in the entire bioluminescent cycle of firefly. The current theoretical calculation could inspire the study on LH2 storage and release of other bioluminescent organisms.
Assuntos
Vaga-Lumes , Luciferina de Vaga-Lumes , Aminoácidos , Animais , Luciferina de Vaga-Lumes/química , Luciferases de Vaga-Lume/metabolismo , Luciferinas , Medições Luminescentes/métodos , Modelos TeóricosRESUMO
Our therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CLPro of coronaviruses, so that the luminescent biosensor is turned on upon 3CLPro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development.
Assuntos
Antivirais/isolamento & purificação , Técnicas Biossensoriais/métodos , Proteases 3C de Coronavírus/metabolismo , SARS-CoV-2/fisiologia , Replicação Viral , Animais , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Células HEK293 , Humanos , Luciferases de Vaga-Lume/metabolismo , Mucosa Nasal/virologia , Pirazolonas/farmacologia , Piridonas/farmacologia , SARS-CoV-2/metabolismo , Células Vero , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
We have earlier shown that p53-FL and its translational isoform ∆40p53 are differentially regulated. In this study, we have investigated the cellular effect of ∆40p53 regulation on downstream gene expression, specifically miRNAs. Interestingly, ∆40p53 showed antagonistic regulation of miR-186-5p as compared to either p53 alone or a combination of both the isoforms. We have elucidated the miR-186-5p mediated effect of ∆40p53 in cell proliferation. Upon expression of ∆40p53, we observed a significant decrease in YY1 levels, an established target of miR-186-5p, which is involved in cell proliferation. Further assays with anti-miR-186 established the interdependence of ∆40p53- miR-186-5p-YY1- cell proliferation. The results unravel a new dimension toward the understanding of ∆40p53 functions, which seems to regulate cellular fate independent of p53FL.
Assuntos
Proliferação de Células/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo , Células HCT116 , Humanos , Luciferases de Vaga-Lume , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fator de Transcrição YY1/antagonistas & inibidoresRESUMO
CONTEXT: The natural products derived from Capparis ecuadorica H.H. Iltis (Capparaceae) could have great potential for anti-inflammation since they inhibited the inflammatory response in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. OBJECT: This study investigated the anti-inflammatory effects and related mechanism of methanol extract of C. ecuadorica leaves (MCE) during atopic dermatitis (AD) responses. MATERIALS AND METHODS: Alterations in the phenotypical markers for AD, luciferase signal, iNOS-mediated COX-2 induction pathway, and inflammasome activation were analysed in non-Tg (n = 5) and 15% phthalic anhydride (PA) treated IL-4/Luc/CNS-1 transgenic (Tg) HR1 mice (n = 5 per group), subsequent to treatment with acetone-olive oil (AOO), vehicle (DMSO) and two dose MCE (20 and 40 mg/kg) three times a week for 4 weeks. RESULTS: MCE treatment reduced the intracellular ROS level (48.2%), NO concentration (7.1 mmol/L) and inflammatory cytokine expressions (39.1%) in the LPS-stimulated RAW264.7 cells. A significant decrease was detected for ear thickness (16.9%), weight of lymph node (0.7 mg), IgE concentration (1.9 µg/mL), and epidermal thickness (31.8%) of the PA + MCE treated Tg mice. MCE treatment induced the decrease of luciferase signal derived from the IL-4 promoter and the recovery of the IL-4 downstream regulator cytokines. PA + MCE treated Tg mice showed decreasing infiltration of mast cells (42.5%), iNOS-mediated COX-2 induction pathway, MAPK signalling pathway and inflammasome activation in the ear tissue. CONCLUSIONS: These findings provide the first evidence that MCE may have great potential to suppress chemical-induced skin inflammation through the suppression of IL-4 cytokine and the iNOS-mediated COX-2 induction pathway, and activation of inflammasome.
Assuntos
Anti-Inflamatórios/farmacologia , Capparis , Dermatite Atópica/tratamento farmacológico , Interleucina-4/genética , Luciferases de Vaga-Lume/genética , Anidridos Ftálicos/toxicidade , Extratos Vegetais/farmacologia , Animais , Ciclo-Oxigenase 2/fisiologia , Dermatite Atópica/induzido quimicamente , Inflamassomos/fisiologia , Mastócitos/fisiologia , Camundongos , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo II/fisiologia , Células RAW 264.7RESUMO
HSP70 is a powerful antiapoptotic protein that can block the extrinsic and intrinsic pathways of apoptosis. The present study describes a rapid, sensitive, and inexpensive system using luciferase as a reporter for the functional analysis of apoptotic compounds. For this approach, the co-transformation of Escherichia coli cells was performed with two expression vectors containing Hsp70 and firefly luciferase. It was found that the luciferase inactivated by heat treatment (40-46 °C for 10 min) was approximately reactivated at room temperature and regained 70% of its initial activity before heat inactivation after 60 min. The results show that the reactivation of thermally inactivated luciferase was inhibited in living cells by treatment with VER-155008 and pifitrin-µ as Hsp70 inhibitors, with half-maximal inhibitory concentration of 124 and 384 µM, respectively. The sensitivity of this method for detecting VER-155008 and pifitrin-µ was about 8 and 25 µM, respectively. Also, this reporter system showed no response to doxorubicin and dactinomycin, which bind to DNA, and we used these anticancer compounds as control compounds. Therefore, for the first time, a rapid and simple real-time system using luciferase as a reporter is introduced for the screening of apoptosis-inducing compounds based on suppression of Hsp70 in E. coli cells.
Assuntos
Apoptose/efeitos dos fármacos , Genes Reporter , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Luciferases de Vaga-Lume/genética , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Escherichia coli/genética , Proteínas de Choque Térmico HSP70/genéticaRESUMO
The therapeutic potential of microRNA-21 (miR-21) small-molecule inhibitors has been of particular interest to medicinal chemists. Moreover, the development of more facile screening methods is lacking. In the present study, two potential screening strategies for miR-21 small-molecule inhibitor including the stem-loop reverse transcription-quantitative PCR and dual luciferase reporter assay system were demonstrated and discussed in detail. A pmirGLO-miR21cswt plasmid and its two different mutants were constructed for dual luciferase reporter assay system. In addition, the sensitivity and specificity of these two methods were validated. Our results demonstrated that both strategies are decent choices for the screening of small-molecule inhibitors for miR-21 and possibly other miRNAs. Eventually, we applied our optimized strategy to discover and characterize several promising compounds such as azobenzene derivate A, enoxacin, and norfloxacin for their potential impact on intracellular miR-21 concentration.
Assuntos
Genes Reporter/efeitos dos fármacos , Luciferases de Vaga-Lume/antagonistas & inibidores , MicroRNAs/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Bibliotecas de Moléculas Pequenas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/genética , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Células Tumorais CultivadasRESUMO
The present manuscript describes a novel bioassay consisting of apyrase and heat shock protein 90 (Hsp90) without additional co-chaperone supplementation; intended for high-throughput screening of anti-cancer drugs and prognosis of stress. In this regard, Hsp90 and adenosine 5'-triphosphate (ATP) mediated firefly luciferase (FLuc) kinetics was investigated using apyrase and FLuc as client proteins. Bioluminescent assay containing Hsp90, ATP, and apyrase led to complete loss of luminescence at 50⯰C which indicates the protective role of Hsp90 against thermal denaturation. Similarly, the assay sample comprising Hsp90, ATP, and FLuc showed 2 fold increments in luminescence than their counterparts. Introduction of bovine serum albumin (BSA) to the pre-incubated assay mixture led to an initial rise in the luminescence (28%) in comparison to the sample containing Hsp90, ATP and FLuc. Therefore, FLuc based HTS assays are not suitable for clinical samples which may contain stabilizing agents. However, thermally denatured FLuc and apyrase could not regain their active conformation even when Hsp90 and ATP were introduced in the assay system. This observation justifies the role of Hsp90 to be protective rather than a reparation agent when acts without co-chaperones.
Assuntos
Trifosfato de Adenosina/metabolismo , Apirase/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Luciferases de Vaga-Lume/metabolismo , Ativação Enzimática , Cinética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Dobramento de Proteína , Redobramento de Proteína , TemperaturaRESUMO
Conventional photodynamic therapy is severely constrained by the limited light-penetration depth in tissue. Here, we show efficient photodynamic therapy (PDT) mediated by bioluminescence resonance energy transfer (BRET) that overcomes the light-penetration limitation. The photosensitizer Rose Bengal (RB) was loaded in biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles, which were then conjugated with firefly luciferase. Spectroscopic characterizations indicated that BRET effectively activated RB to generate reactive oxygen species (ROS). In vitro studies of the cellular cytotoxicity and photodynamic effect indicated that cancer cells were effectively destroyed by BRET-PDT treatment. In vivo studies in a tumor-bearing mouse model demonstrated that tumor growth was significantly inhibited by BRET-PDT in the absence of external light irradiation. The BRET-mediated phototherapy provides a promising approach to overcome the light-penetration limitation in photodynamic treatment of deep-seated tumors.
Assuntos
Materiais Biocompatíveis , Transferência de Energia , Nanopartículas/química , Fotoquimioterapia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/efeitos adversos , Animais , Linhagem Celular Tumoral , Humanos , Luciferases de Vaga-Lume/metabolismo , Luminescência , Camundongos , Camundongos Endogâmicos ICR , Espécies Reativas de Oxigênio/metabolismoRESUMO
Glioblastoma (GBM) is the most aggressive and lethal form of brain cancer. Standard therapies are non-specific and often of limited effectiveness; thus, efforts are underway to uncover novel, unorthodox therapies against GBM. In previous studies, we investigated Withaferin A, a steroidal lactone from Ayurvedic medicine that inhibits proliferation in cancers including GBM. Another novel approach, tumor treating fields (TTFields), is thought to disrupt mitotic spindle formation and stymie proliferation of actively dividing cells. We hypothesized that combining TTFields with Withaferin A would synergistically inhibit proliferation in glioblastoma. Human glioblastoma cells (GBM2, GBM39, U87-MG) and human breast adenocarcinoma cells (MDA-MB-231) were isolated from primary tumors. The glioma cell lines were genetically engineered to express firefly luciferase. Proliferative potential was assessed either by bioluminescence imaging or cell counting via hemocytometer. TTFields (4 V/cm) significantly inhibited growth of the four cancer cell lines tested (n = 3 experiments per time point, four measurements per sample, p < 0.02 at least; 2-way ANOVA, control vs. treatment). The combination of Withaferin A (10-100 nM) with TTFields significantly inhibited the growth of the glioma cells to a degree beyond that of Withaferin A or TTFields alone. The interaction of the Withaferin A and TTFields on glioma cells was found to be synergistic in nature (p < 0.01, n = 3 experiments). These findings were validated by both bioluminescence and hemocytometric measurements. The combination of Withaferin A with TTFields represents a novel approach to treat GBM in a manner that is likely better than either treatment alone and that is synergistic.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/terapia , Proliferação de Células , Terapia por Estimulação Elétrica , Glioma/terapia , Vitanolídeos/uso terapêutico , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Adenocarcinoma/terapia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/fisiopatologia , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Terapia Combinada , Doxorrubicina/uso terapêutico , Terapia por Estimulação Elétrica/métodos , Glioma/patologia , Glioma/fisiopatologia , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , TemperaturaRESUMO
Cellular MRI involves sensitive visualization of iron-labeled cells in vivo but cannot differentiate between dead and viable cells. Bioluminescence imaging (BLI) measures cellular viability, and thus we explored combining these tools to provide a more holistic view of metastatic cancer cell fate in mice. Human breast carcinoma cells stably expressing Firefly luciferase were loaded with iron particles, injected into the left ventricle, and BLI and MRI were performed on days 0, 8, 21 and 28. The number of brain MR signal voids (i.e., iron-loaded cells) on day 0 significantly correlated with BLI signal. Both BLI and MRI signals decreased from day 0 to day 8, indicating a loss of viable cells rather than a loss of iron label. Total brain MR tumour volume on day 28 also correlated with BLI signal. Overall, BLI complemented our sensitive cellular MRI technologies well, allowing us for the first time to screen animals for successful injections, and, in addition to MR measures of cell arrest and tumor burden, provided longitudinal measures of cancer cell viability in individual animals. We predict this novel multimodality molecular imaging framework will be useful for evaluating the efficacy of emerging anti-cancer drugs at different stages of the metastatic cascade.
Assuntos
Neoplasias Encefálicas/patologia , Nanopartículas de Magnetita/química , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Imageamento por Ressonância Magnética , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Transplante Heterólogo , Carga TumoralRESUMO
Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic ß-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Regiões Promotoras Genéticas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , gama-Globinas/genética , Animais , Linhagem Celular , Vaga-Lumes/genética , Humanos , Luciferases de Vaga-Lume/genética , Luciferases de Renilla/genética , Renilla/genética , Transfecção/métodosRESUMO
Luciferase-dependent assays, important for biochemical analyses of cytotoxicity and reporter genes, may be perturbed by compounds interfering with the luciferase reaction. We analyzed the impact of different aluminum (Al) species on a luciferase-based assay for determination of cellular adenosine triphosphate. Al(0) nanoparticles (Al(0)-NPs) but not Al2O3-NPs decreased luminescence, correlated to high absorbance of Al(0)-NPs. By contrast, Al ions increased the luminescent signal. Data demonstrate that luciferase-dependent assays can be reciprocally disturbed by Al-NPs or Al ions in a specific manner, depending on the particular Al species. Careful interpretation of data from such experiments is essential in order to obtain conclusive results.
Assuntos
Óxido de Alumínio/farmacologia , Alumínio/farmacologia , Bioensaio , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Trifosfato de Adenosina/análise , Alumínio/química , Alumínio/metabolismo , Óxido de Alumínio/química , Óxido de Alumínio/metabolismo , Apoptose/efeitos dos fármacos , Células CACO-2 , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Luciferases de Vaga-Lume/genética , Nanopartículas/química , Nanopartículas/metabolismo , Relação Estrutura-AtividadeRESUMO
A Pseudorabies virus (PRV) variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. However, identification of single guide RNA (sgRNA) through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP) as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures.
Assuntos
Antivirais/farmacologia , Sistemas CRISPR-Cas , Avaliação Pré-Clínica de Medicamentos/métodos , Expressão Gênica , Vetores Genéticos , Herpesvirus Suídeo 1/efeitos dos fármacos , Luciferases de Vaga-Lume/genética , RNA Guia de Cinetoplastídeos , Animais , Linhagem Celular , Ordem dos Genes , Genes Reporter , Vetores Genéticos/genética , Herpesvirus Suídeo 1/genética , Replicação Viral/efeitos dos fármacosRESUMO
Photodynamic therapy (PDT) of cancer is known for its limited number of side effects, and requires light, oxygen and photosensitizer. However, PDT is limited by poor penetration of light into deeply localized tissues, and the use of external light sources is required. Thus, researchers have been studying ways to improve the effectiveness of this phototherapy and expand it for the treatment of the deepest cancers, by using chemiluminescent or bioluminescent formulations to excite the photosensitizer by intracellular generation of light. The aim of this Minireview is to give a précis of the most important general chemi-/bioluminescence mechanisms and to analyze several studies that apply them for PDT. These studies have demonstrated the potential of utilizing chemi-/bioluminescence as excitation source in the PDT of cancer, besides combining new approaches to overcome the limitations of this mode of treatment.
Assuntos
Neoplasias/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/uso terapêutico , Animais , Luciferina de Vaga-Lumes/metabolismo , Humanos , Imidazóis/metabolismo , Luciferases de Vaga-Lume/metabolismo , Luciferases de Renilla/metabolismo , Luminescência , Substâncias Luminescentes/metabolismo , Luminol/metabolismo , Neoplasias/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Pirazinas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Interleukin (IL)-4 acts on T cells as a growth and activation factor, and promotes the differentiation of type 2 T helper cells. In T cells, expression of the gene encoding IL-4 is regulated by inducible or constitutive factors. Yin-Yang (YY)-1 is one of constitutive transcription factors binding to the IL-4 promoter. The recently identified YY2 protein is similar to YY1, with both sharing high levels of homology in their zinc finger motifs. However, the role of YY2 in T cells is unclear. YY1 and YY2 were constitutively expressed in EL4 T cells, and their expression was not dependent on stimulation. IL-4 promoter (-741/+56 fragment) activity was enhanced by YY1, but inhibited by YY2. The enhanced IL-4 promoter activity by YY1 was reduced by simultaneous expression of YY2. In addition, the DNA binding affinity of YY1 to the IL-4 promoter was adversely affected by YY2. Our results suggest that YY1 and YY2 exert opposing effects on the IL-4 promoter as they compete for the same DNA binding sites.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Regulação da Expressão Gênica , Interleucina-4/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Interleucina-4/metabolismo , Luciferases de Vaga-Lume/genética , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Transfecção , Fator de Transcrição YY1/genética , Dedos de Zinco/genéticaRESUMO
The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode's ATP levels.(1-3) Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.(4,5) In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Medições Luminescentes/métodos , Mitocôndrias/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Inibidores Enzimáticos/farmacologia , Luciferases de Vaga-Lume/antagonistas & inibidores , Mitocôndrias/fisiologia , Ácido Oxaloacético/farmacologia , Paraquat/farmacologia , Rotenona/farmacologia , Desacopladores/farmacologiaRESUMO
Chronic inflammation underlies the pathological progression of various diseases, and thus many efforts have been made to quantitatively evaluate the inflammatory status of the diseases. In this study, we generated a highly sensitive inflammation-monitoring mouse system using a bacterial artificial chromosome (BAC) clone containing extended flanking sequences of the human interleukin 6 gene (hIL6) locus, in which the luciferase (Luc) reporter gene is integrated (hIL6-BAC-Luc). We successfully monitored lipopolysaccharide-induced systemic inflammation in various tissues of the hIL6-BAC-Luc mice using an in vivo bioluminescence imaging system. When two chronic inflammatory disease models, i.e., a genetic model of atopic dermatitis and a model of experimental autoimmune encephalomyelitis (EAE), were applied to the hIL6-BAC-Luc mice, luciferase bioluminescence was specifically detected in the atopic skin lesion and central nervous system, respectively. Moreover, the Luc activities correlated well with the disease severity. Nrf2 is a master transcription factor that regulates antioxidative and detoxification enzyme genes. Upon EAE induction, the Nrf2-deficient mice crossed with the hIL6-BAC-Luc mice exhibited enhanced neurological symptoms concomitantly with robust luciferase luminescence in the neuronal tissue. Thus, whole-body in vivo monitoring using the hIL6-BAC-Luc transgenic system (WIM-6 system) provides a new and powerful diagnostic tool for real-time in vivo monitoring of inflammatory status in multiple different disease models.
Assuntos
Interleucina-6/genética , Imagem Corporal Total , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Encéfalo/imunologia , Encéfalo/metabolismo , Células Cultivadas , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/imunologia , Dermatite Atópica/metabolismo , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Genes Reporter , Humanos , Interleucina-6/biossíntese , Lipopolissacarídeos/farmacologia , Luciferases de Vaga-Lume/biossíntese , Luciferases de Vaga-Lume/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Especificidade de Órgãos , Sepse/imunologia , Sepse/metabolismoRESUMO
Cochinchinones M-U (1-9), together with 12 known compounds (10-21), were isolated from the stems of Cratoxylum cochinchinense (Lour.) Blume. Their structures were determined on the basis of extensive spectroscopic data analyses. In addition, their retinoid X receptor-α transcriptional activities were evaluated using an in vitro assay.