Cyclosporin A Inhibits Calcineurin/Nuclear Factor of Activated T-Cells Signaling and Induces Apoptosis in Retinoblastoma Cells

Abstract

Although the clinical efficacy of cyclosporin A (CSA) in retinoblastoma (RB) has been attributed to multidrug resistance reversal activity, the authors hypothesized that CSA is also directly toxic to RB cells through inhibition of calcineurin (CN)/nuclear factor of activated T-cells (NFAT) signaling. Antiproliferative effects of CSA, PSC-833 (a CSA analogue that does not inhibit CN), and FK506 (a CN inhibitor structurally unrelated to CSA) were evaluated in Y79 and Weri-RB1 cells by WST-1 assay. Apoptosis induction by CSA and PSC-833 was measured by detection of caspase 3/7 activity and by flow cytometry, using annexin-V and 7-AAD stains. Expression of CN was assayed in RB cells by immunocytochemistry. Expression of NFAT, a CN-dependent transcription factor family, and FK506 binding protein 12/12.6 (FKBP12/12.6), effectors of CN inhibition by FK506, was assayed in RB cells by Western blot analysis. NFAT activity was assayed in CSA-treated and -untreated Y79 cells transfected with an NFAT-sensitive reporter gene. CSA induced dose-dependent antiproliferative and proapoptotic effects at clinically achievable levels in Y79 and Weri-RB1 cells. PSC-833 induced antiproliferative effects only at nonphysiologic concentrations with minimal associated apoptosis. FK506 induced minimal antiproliferative effects in RB cell lines, probably due to trace or absent FKBP12/12.6 expression. RB cell lines expressed CN-alpha, CN-beta, NFATc1, and NFATc3. CSA treatment also potently inhibited NFAT-mediated reporter gene transcription. These results demonstrate functional integrity of the CN/NFAT signaling cascade in RB cells and suggest that CSA is cytotoxic to RB cells through inhibition of this pathway and consequent apoptosis induction.