Abstract
Abstract: :
Purpose: Cyclosporin A (CSA) is clinically effective in the treatment of advanced retinoblastoma (RB) when administered in combination with carboplatin, etoposide, and vincristine. While the therapeutic utility of CSA has been attributed to multidrug–resistance (MDR) reversal activity, we investigated the hypothesis that CSA might also be directly toxic to RB cells through inhibition of calcineurin (CN)/nuclear factor of activated T–cells (NFAT) signaling. Methods: Dose–dependent anti–proliferative 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 human RB cells in vitro by WST–1 assay. In addition, apoptosis induction by CSA and PSC–833 was measured by detection of caspase 3/7 activity, and the frequency of apoptotic cells was quantified by flow cytometry using a combination of Annexin V and 7–AAD stains. Expression of the α and ß subunits of CN in RB cells was assayed by immunocytochemical staining. Expression of NFAT, a CN–dependent transcription factor family, and FK506 binding protein 12 (FKBP12), a critical effector of CN inhibition by FK506, was assayed in RB whole cell lysates by immunoprecipitation and Western blot analysis. Lastly, NFAT activity was measured in CSA–treated and –untreated Y79 cells transfected with an NFAT–sensitive reporter gene. Results: CSA induced dose–dependent anti–proliferative and pro–apoptotic effects at clinically achievable levels in Y79 and Weri–RB1 cells, whereas PSC–833 induced dose–dependent anti–proliferative effects only at high, non–physiologic concentrations with minimal evidence of apoptosis. Consistent with these results, both cell lines expressed high levels of CN, NFATc1, and NFATc3, and CSA treatment potently inhibited NFAT–mediated transcription in our transfection assay. Unexpectedly, FK506 induced no significant anti–proliferative effects, which we attribute to our finding of absent or minimal expression of FKBP12 in these cell lines. Conclusions: Our results demonstrate the 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 induction of apoptosis. Therefore, CSA may provide therapeutic benefits in the treatment of RB independent of its MDR reversal activity, and strategic use of CSA may improve treatment efficacy and reduce patient exposure to chemotherapeutic agents.
Keywords: retinoblastoma • oncology • cyclosporine