Cyclosporin a inhibits calcineurin/nuclear factor of activated T-cells signaling and induces apoptosis in retinoblastoma cells.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Eckstein
Lauren A
LA
Ocular Oncology Unit, Department of Ophthalmology, University of California at San Francisco, 10 Koret Way, San Francisco, CA 94143, USA.
Van Quill
Kurtis R
KR
Bui
Steven K
SK
Uusitalo
Marita S
MS
O'Brien
Joan M
JM
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D019703:Calcineurin / Q000235:genetics; D065095:Calcineurin Inhibitors; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology*; D003524:Cyclosporins / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008213:Lymphocyte Activation / Q000187:drug effects; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D019572:Retinal Neoplasms / Q000378:metabolism / Q000473:pathology*; D012175:Retinoblastoma / Q000378:metabolism / Q000473:pathology*; D015398:Signal Transduction / Q000187:drug effects; D013601:T-Lymphocytes / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics; D014407:Tumor Cells, Cultured
D065095:Calcineurin Inhibitors; D003524:Cyclosporins; D004268:DNA-Binding Proteins; D007166:Immunosuppressive Agents; D050778:NFATC Transcription Factors; C494003:NFATC1 protein, human; C494008:NFATC3 protein, human; D009687:Nuclear Proteins; D014157:Transcription Factors; D016572:Cyclosporine; D019703:Calcineurin; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D022061:Tacrolimus Binding Protein 1A; C070272:valspodar; D016559:Tacrolimus