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Shahar Frenkel, Dudi Shneor, Alik Honigman, Jacob Pe'er; NFkB Inhibition in Uveal Melanoma - construction of a new delivery system. Invest. Ophthalmol. Vis. Sci. 2014;55(13):846. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously shown that NFkB inhibition leads to diminished uveal melanoma cell viability in vitro, and to necrosis of tumors in vivo in the mouse intrahepatic direct injection model. Here we describe the design of a targeted delivery system which affects only dividing cells - a recombinant Murine leukemia virus (MuLV)-based replication-competent retroviruses (RCR) delivery system.
We exchanged the GFP sequence in the IRES-GFP cassette of the MuLV-based RCR pACE with the NFkB nuclear localization sequence VQRKRQKLMP (pACE-NFkB-NLS). Five uveal melanoma cell lines were infected with the pACE-NFkB-NLS vector. Infected cells were grown on “chamber-slides” without or with the TNFα activator of NFkB, and the subcellular localization of NFkB was analyzed with a laser scanning confocal microscope (Nikon). Activity of NFkB was analyzed by transfecting infected cell with a luciferase based reporter gene. Cell viability and caspase 3 activity were determined using the Fluorescent Cell Viability and Caspase-Glo 3/7 Assays (Promega).
Expression of the NLS peptide inside the cells created a competition with the activated NFkB, and reduced its nuclear localization and its ability to trans-activate target genes.
Inhibition of NFkB reduces cell viability and increases apoptosis. This new modality for NFkB inhibition which can spread through solid tumors is expected to better affect tumors in vivo than IV/IP administration of chemotherapeutics.
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