Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Apr 15:873:172981.
doi: 10.1016/j.ejphar.2020.172981. Epub 2020 Jan 31.

In vitro benchmarking of NF-κB inhibitors

Alexandria P Harrold  1 , Megan M Cleary  1 , Narendra Bharathy  1 , Melvin Lathara  2 , Noah E Berlow  1 , Nicholas K Foreman  3 , Andrew M Donson  3 , Vladimir Amani  3 , William J Zuercher  4 , Charles Keller  5
Affiliations

In vitro benchmarking of NF-κB inhibitors

Alexandria P Harrold et al. Eur J Pharmacol. .

Abstract

Dysregulated activity of the transcription factors of the nuclear factor κb (NF-κB) family has been implicated in numerous cancer types, inflammatory diseases, autoimmune disease, and other disorders. As such, selective NF-κB pathway inhibition is an attractive target to researchers for preclinical and clinical drug development. A plethora of commercially and clinically available inhibitors claim to be NF-κB specific; however, such claims of specificity are rarely quantitative or benchmarked, making the biomedical literature difficult to contextualize. This imprecision is worsened because some NF-κB reporter systems have low signal-to-noise ratios. Herein, we use a robust, defined, commercially available reporter system to benchmark NF-κB agonists and antagonists for the field. We also functionally characterize a RELA fusion-positive ependymoma cell culture with validated NF-κB inhibitor compounds.

Keywords: Ependymoma; NF-κB; RELA.

PubMed Disclaimer

Conflict of interest statement

Declaration of competing interest The authors have no competing interests.

Figures

Fig 1.
Fig 1.. Examination of NFκB antagonists in HEK293 cells.
Inhibition of NF-κB signaling was examined in HEK293 cells that stably express a NF-κB luciferase reporter and were graphed versus a measurement of cell viability by a non-luciferase luminescence assay (CTG). Cells were stimulated with TNFα and treated with either antagonist or vehicle (DMSO) for 24 h. Graphs are representative of a single experiment where each condition was performed in triplicate. Each experiment was repeated 3 times. Mean ± S.E.M.
Fig 2.
Fig 2.. Examination of NF-κB agonists in HEK293 cells.
NF-κB signaling was examined in HEK293 cells that stably express a NF-κB luciferase reporter and were graphed versus a measurement of cell viability by a non-luciferase luminescence assay (CTG). Cells were treated with either antagonist or vehicle (DMSO) for 24 h. Graphs are representative of a single experiment where each condition was performed in triplicate. Each experiment was repeated 3 times. Mean ± S.E.M.
Fig 3.
Fig 3.. Characterization of MAF1329 ependymoma cells.
(A) Circos plot depiction of tumor genome MAF1329 (B) Western blot analysis confirmed presence of wild type RELA as well as C11orf95-RELA fusion protein in ependymoma primary cell culture MAF1329. (C) Cell viability of MAF1329 cells treated with NF-κB antagonists specifically targeting IκBα phosphorylation.
See this image and copyright information in PMC

References

    1. Ashikawa K, Majumdar S, Banerjee S, Bharti AC, Shishodia S, Aggarwal BB, 2002. Piceatannol inhibits TNF-induced NF-kappaB activation and NF-kappaB-mediated gene expression through suppression of IkappaBalpha kinase and p65 phosphorylation. J. Immunol 169, 6490–7. - PubMed
    1. Baeuerle PA, Baltimore D, 1988. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-κB transcription factor. Cell 53, 211–217. 10.1016/0092-8674(88)90382-0 - DOI - PubMed
    1. Birrell MA, Hardaker E, Wong S, Mccluskie K, Catley M, De Alba J, Newton R, Haj-Yahia S, Tao K, Watts CJ, Shaw RJ, Savage TJ, Belvisi MG, Belvisi G, 2005. I-B Kinase-2 Inhibitor Blocks Inflammation in Human Airway Smooth Muscle and a Rat Model of Asthma. Am J Respir Crit Care Med 172, 962–971. 10.1164/rccm.200412-1647OC - DOI - PubMed
    1. Bortoluzzi A, Vincenzi F, Govoni M, Padovan M, Ravani A, Borea PA, Varani K, 2016. A2Aadenosine receptor upregulation correlates with disease activity in patients with systemic lupus erythematosus. Arthritis Res. Ther 18. 10.1186/s13075-016-1089-8 - DOI - PMC - PubMed
    1. Brown K, Gerstberger S, Carlson L, Franzoso G, Siebenlist U, n.d. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. Sci. 1995 267, 1485–1488. - PubMed