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C.L. Dalgard, J. Tsui, K.R. Van Quill, J.M. O'Brien; Selective Inhibiton of Calcineurin / Nuclear Factor of Activated T–Cells Signaling by the Cell Permeable Peptide 11R–VIVIT is Insufficient for Induction of Apoptosis in Retinoblastoma Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2808.
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© ARVO (1962-2015); The Authors (2016-present)
We previously demonstrated that Cyclosporin A (CsA) induces antiproliferative and proapoptotic effects in retinoblastoma (RB) cells, and that these effects are correlated with potent inhibition of NFAT (nuclear factor of activated T cells) signaling and NFAT–mediated reporter gene transcription. These results suggested that the cytotoxic effects of CsA in RB cells are mediated by inhibition of the NFAT signaling pathway. On the basis of this hypothesis we tested 11R–VIVIT, a cell–permeable inhibitor of NFAT, in RB cells. We considered that this selective agent could provide a directed therapeutic strategy for RB patients who are prone to second tumor development.
Y79 and WERI–Rb1 human retinoblastoma cells were treated with 11R–VIVIT at concentrations up to 2 µM. After 72 hours, cell viability was assayed by WST–1 Cell Proliferation Reagent (Roche). Total RNA was also isolated by the use of RNeasy Mini Kit (Qiagen) and gene expression of NFAT target genes were determined by qPCR on the MyIQ Real–Time PCR Detection System (Bio–Rad). The activation of apoptosis was assayed through the use of Caspase–Glo 3/7 Assay (Promega). The effect of 11R–VIVIT on CsA–mediated cytotoxicity was also examined. Y79 and WERI–Rb1 cells were treated with 1 µM 11R–VIVIT and concentrations of CsA up to 50 µg/mL. After 72 and 96 hours, cell viability and caspase 3/7 activity was determined.
11R–VIVIT did not induce antiproliferative or proapoptotic effects in RB cells at concentrations tested. A 5–fold and 4–fold decrease in gene expression of NFAT target genes (TNF–a and COX–2, respectively) was found in Y79 cells treated with 1 µM 11R–VIVIT for 72 hours, relative to controls. 11R–VIVIT did significantly decreased cell viability in RB cells treated with CsA. IC50 of CsA in Y79 cells treated with 11R–VIVIT was 22 µg/mL compared to 28 µg/mL in controls without CsA treatment.
Selective blockade of the NFAT signaling pathway by 11R–VIVIT is insufficient to induce cytotoxicity in RB cells. These results suggest that CsA does not mediate its cytotoxic effects solely through inhibition of calcineurin / NFAT signaling. However, since 11R–VIVIT does potentiate cytotoxicity mediated by CsA, the NFAT pathway may be important for cell survival and inhibition of apoptosis in RB cells. These findings call for additional investigation into the mechanisms of CsA–mediated cytotoxicity in RB cells.
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