Establishment of a Cell-Based Assay to Screen Regulators of the Hypoxia-Inducible Factor-1-Dependent Vascular Endothelial Growth Factor Promoter

Ji, Deng Bo; Zhu, Hai; Jia, Yali; Li, Chang Ling

doi:10.1248/bpb.31.2255

Cited by 4 publications

(4 citation statements)

References 16 publications

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“…The Prestwick Chemical Library consists of 1200 small compounds with high chemical and pharmacological diversity. The number of hits as percentage of the library was 1.5%, comparable with other published screens, 29–31 suggesting that the assay was specific and would yield a low number of false-positives. Nonetheless, only 11 of the initial 18 hits were confirmed in the dose–response analyses, and this was expected owing to experimental variation.…”

Section: Discussionsupporting

confidence: 82%

Boosting innate immunity: Development and validation of a cell-based screening assay to identify LL-37 inducers

et al. 2013

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Innate immunity, the front line of our defence against pathogens, relies, to a great extent, on the production of antimicrobial peptides (AMPs). These peptides exhibit antimicrobial activity and immunomodulatory properties. In humans, AMPs include the defensins (α- and β-families) and the cathelicidin, LL-37. Bacterial resistance against antibiotics is a growing concern, and novel antimicrobial strategies are needed urgently. Hence, the concept of strengthening immune defences against infectious microbes by inducing AMP expression may represent novel or complementary pharmaceutical interventions in the treatment or prevention of infections. We have developed and validated a robust cell-based reporter assay for LL-37 expression, which serves as a marker for a healthy epithelial barrier. This reporter assay can be a powerful tool for high-throughput screenings. We first employed our assay to screen a panel of histone deacetylase inhibitors and derivatives, and then the Prestwick Chemical Library of Food and Drug Administration-approved compounds. After hit confirmation and independent validation in the parental cell line we identified five novel inducers of LL-37. This reporter assay will help to identify novel drug candidates for the treatment and prevention of infections. Importantly, the pattern of hits obtained may suggest cellular pathways and key mediators involved in the regulation of AMP expression.

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Section: Discussionsupporting

confidence: 82%

Boosting innate immunity: Development and validation of a cell-based screening assay to identify LL-37 inducers

et al. 2013

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“…The number of hits as a percentage of the library (1.8%) was comparable to other published screens [36], [37], [38], suggesting that the assay was specific with a low number of false positives. Nevertheless, we had set the initial screen threshold of 20% upregulation low enough to avoid false negatives, in the expectation that some of the initial hits would be false positives due to ‘statistical noise’.…”

Section: Discussionsupporting

confidence: 77%

Drug Discovery for Duchenne Muscular Dystrophy via Utrophin Promoter Activation Screening

et al. 2011

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BackgroundDuchenne muscular dystrophy (DMD) is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use.Methodology/Principal FindingsWe developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells.Conclusions/SignificanceWe have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD.

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“…Chemicals that alter gene expression or protein-protein interaction can be detected by immunofluorescence or intracellular reporter gene assays. A battery of such assays have been applied to screen and identify chemicals targeting cellular signal pathways including HIFs (180,182,183,361), NF-κB ( 184), IL-6 (181), IL-8 (185) and TGFs (186,187).…”

Section: In Vitro and In Vivo Angiogenesis Assays Including Hts Assaymentioning

confidence: 99%

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: focus on the cancer hallmark of tumor angiogenesis

Hu¹,

Brooks

Dormoy

et al. 2015

CARCIN

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One of the important 'hallmarks' of cancer is angiogenesis, which is the process of formation of new blood vessels that are necessary for tumor expansion, invasion and metastasis. Under normal physiological conditions, angiogenesis is well balanced and controlled by endogenous proangiogenic factors and antiangiogenic factors. However, factors produced by cancer cells, cancer stem cells and other cell types in the tumor stroma can disrupt the balance so that the tumor microenvironment favors tumor angiogenesis. These factors include vascular endothelial growth factor, endothelial tissue factor and other membrane bound receptors that mediate multiple intracellular signaling pathways that contribute to tumor angiogenesis. Though environmental exposures to certain chemicals have been found to initiate and promote tumor development, the role of these exposures (particularly to low doses of multiple substances), is largely unknown in relation to tumor angiogenesis. This review summarizes the evidence of the role of environmental chemical bioactivity and exposure in tumor angiogenesis and carcinogenesis. We identify a number of ubiquitous (prototypical) chemicals with disruptive potential that may warrant further investigation given their selectivity for high-throughput screening assay targets associated with proangiogenic pathways. We also consider the cross-hallmark relationships of a number of important angiogenic pathway targets with other cancer hallmarks and we make recommendations for future research. Understanding of the role of low-dose exposure of chemicals with disruptive potential could help us refine our approach to cancer risk assessment, and may ultimately aid in preventing cancer by reducing or eliminating exposures to synergistic mixtures of chemicals with carcinogenic potential.

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Establishment of a Cell-Based Assay to Screen Regulators of the Hypoxia-Inducible Factor-1-Dependent Vascular Endothelial Growth Factor Promoter

Cited by 4 publications

References 16 publications

Boosting innate immunity: Development and validation of a cell-based screening assay to identify LL-37 inducers

Boosting innate immunity: Development and validation of a cell-based screening assay to identify LL-37 inducers

Drug Discovery for Duchenne Muscular Dystrophy via Utrophin Promoter Activation Screening

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: focus on the cancer hallmark of tumor angiogenesis

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