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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)
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.
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.
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).
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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