Eckstein Lauren A, Van Quill Kurtis R, Bui Steven K, Uusitalo Marita S, O'Brien Joan M
Ocular Oncology Unit, Department of Ophthalmology, University of California at San Francisco, 10 Koret Way, San Francisco, CA 94143, USA.
Invest Ophthalmol Vis Sci. 2005 Mar;46(3):782-90. doi: 10.1167/iovs.04-1022.
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.
尽管环孢素A（CSA）在视网膜母细胞瘤（RB）中的临床疗效归因于多药耐药逆转活性，但作者推测CSA还可通过抑制钙调神经磷酸酶（CN）/活化T细胞核因子（NFAT）信号通路对RB细胞产生直接毒性。
通过WST-1检测法评估CSA、PSC-833（一种不抑制CN的CSA类似物）和FK506（一种与CSA结构无关的CN抑制剂）对Y79和Weri-RB1细胞的抗增殖作用。使用膜联蛋白-V和7-氨基放线菌素D染色，通过检测半胱天冬酶3/7活性和流式细胞术来测定CSA和PSC-833诱导的细胞凋亡。通过免疫细胞化学法检测RB细胞中CN的表达。通过蛋白质免疫印迹分析检测RB细胞中NFAT（一种CN依赖性转录因子家族）以及FK506结合蛋白12/12.6（FKBP12/12.6，FK506抑制CN的效应分子）的表达。在转染了NFAT敏感报告基因的经CSA处理和未处理的Y79细胞中检测NFAT活性。
在Y79和Weri-RB1细胞中，CSA在临床可达到的水平上诱导了剂量依赖性的抗增殖和促凋亡作用。PSC-833仅在非生理浓度下诱导抗增殖作用，且相关凋亡极少。FK506在RB细胞系中诱导的抗增殖作用极小，可能是由于FKBP12/12.6表达微量或缺失。RB细胞系表达CN-α、CN-β、NFATc1和NFATc3。CSA处理还强烈抑制了NFAT介导的报告基因转录。
这些结果证明了RB细胞中CN/NFAT信号级联的功能完整性，并表明CSA通过抑制该通路并随之诱导细胞凋亡而对RB细胞具有细胞毒性。
