September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
MDM2 inhibition suppresses NF-κB-mediated inflammation in retinal pigment epithelium cells
Author Affiliations & Notes
  • Biraj Mahato
    Cell Biology and Immunology, University of North Texas Health science Center, Fort Worth, Texas, United States
    Laboratory of Ratinal Rehabilitation, North Texas Eye Research Institute, Fort Worth, Texas, United States
  • Footnotes
    Commercial Relationships   Biraj Mahato, None
  • Footnotes
    Support  R01 EY026201-01
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 247. doi:
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    • Get Citation

      Biraj Mahato; MDM2 inhibition suppresses NF-κB-mediated inflammation in retinal pigment epithelium cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):247.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Our group has shown that Nutlin-3, a MDM2 inhibitor inhibits pathologic retinal endothelial cell proliferation through a p53-dependent mechanism, but its p53-independent functions and effect on retinal pigment epithelial (RPE) cell viability have not been adequately elucidated. TNF-α induced RPE inflammation through NF-κB is a known underlying mechanism of uveitis. Here, we sought to investigate the non-canonical, p53-indendepent function of Nutlin-3, and hypothesized that MDM2 inhibitors possess an anti-inflammatory function benefiting patients suffering from retinal inflammatory diseases.

Methods : For all experiments, ARPE-19 cells were cultured to >95% confluence using standard methodology. In vitro cell viability was assessed using a Hemacytometer and the MTT assay. To assess retinal toxicity in vivo, 200 mM of Nultin-3 or vehicle (DMSO) (n=12 rat eyes per condition) was injected once weekly for four weeks. Retinal toxicity was assessed using histopathology, ERG, SD-OCT, and fundus photography. To assess the anti-inflammatory effect of Nutlin-3, ARPE-19 cells were infected with lenti-virus carrying an NF-κB luciferase reporter.

Results : Nutiln-3 did not demonstrate a statistical difference in cell viability at doses lower than 45 mM (N=3, p>0.5). Rats did not show evidence of retinal toxicity, but some rats did develop cataracts, which may be related to the vehicle or injection technique. ARPE-19 cells were pretreated with Nutlin-3 10 μM (a dose that does not alter RPE cell viability) for 30 minutes to 7 hours, and then stimulated with TNF-α (50 ng/ml) for 24 hours. TNF-α alone induced NF-κB luciferase reporter gene activity, and Nutlin-3 pre-treatment significantly attenuated this TNF-α response (p<0.05).

Conclusions : Based on these experiments, these data suggest that Nutlin-3 does not cause retinal toxicity at doses less than 45 mM in vitro and 200 mM in vivo. Interestingly, Nutlin-3 appears to have a novel p53-indepenent function in RPE cells by inhibiting TNF-α mediated NF-κB RPE inflammation.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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