Combination of vorinostat and flavopiridol is selectively cytotoxic to multidrug-resistant neuroblastoma cell lines with mutant TP53.
Combination of vorinostat and flavopiridol is selectively cytotoxic to multidrug-resistant neuroblastoma cell lines with mutant TP53.
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DOI：
10.1158/1535-7163.mct-10-0562
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发表时间：
2010-12
影响因子：
5.7
通讯作者：
Keshelava N
中科院分区：
文献类型：
--
作者：
Huang JM;Sheard MA;Ji L;Sposto R;Keshelava N
由于p53功能丧失导致神经母细胞瘤产生高水平的耐药性，因此p53非依赖性治疗可能在复发性神经母细胞瘤中具有上级活性。我们在一组多药耐药神经母细胞瘤细胞系中检测了伏立诺他（一种组蛋白脱乙酰酶抑制剂）和flavopiridol（一种泛Cdk抑制剂）的活性，这些细胞系包括野生型（wt）和转录活性TP 53（n = 3）、突变型（mt）和功能丧失型（LOF）TP 53（n = 4）或p14 ARF缺失（n = 1）的细胞系。伏立诺他和夫拉吡醇的组合在具有p53-LOF的细胞系中和在用显性阴性p53质粒稳定转染的克隆中是协同的并且具有显著更大的细胞毒性（p<0.001）。通过流式细胞术进行的细胞周期分析表明，对于具有wt TP 53（SK-N-RA）的细胞系，在16-20小时（37%）显著的细胞周期停滞在G2/M，而具有mt TP 53（CHLA-90）的细胞在6-24小时滑入subG 1（25-40%特异性细胞死亡）。在CHLA-90中用伏立诺他+ flavopiridol组合处理后，通过共聚焦显微镜检测有丝分裂细胞死亡的形态学标志，包括纺锤体形成缺陷和胞质分裂异常。该组合导致G2/M蛋白（细胞周期蛋白B1，Mad 2，MPM 2）的表达减少在两个细胞系与mt TP 53，但不是在那些与wt TP 53。Plk 1的表达在所有处理过的细胞系中均降低。siRNA敲除Mad 2和细胞周期蛋白B1或Plk 1协同降低CHLA-90细胞的克隆形成。HDAC抑制剂和flavopiridol的组合可能是治疗具有p53-LOF的神经母细胞瘤的独特方法，其引起有丝分裂失败的诱导。
As p53-loss of function confers high-level drug resistance in neuroblastoma, p53-independent therapies might have superior activity in recurrent neuroblastoma. We tested the activity of vorinostat, a histone deacetylase inhibitor, and flavopiridol, a pan-Cdk inhibitor, in a panel of multidrug-resistant neuroblastoma cell lines that included lines with wild-type (wt) and transcriptionally active TP53 (n = 3), mutated (mt) and loss of function (LOF) TP53 (n = 4) or p14ARF deletion (n = 1). The combination of vorinostat and flavopiridol was synergistic and significantly more cytotoxic (p<0.001) in cell lines with p53-LOF and in the clones stably transfected with dominant negative p53 plasmids. Cell cycle analysis by flow cytometry demonstrated prominent cell cycle arrest in G2/M for a cell line with wt TP53 (SK-N-RA) at 16–20 hours (37%), while cells with mt TP53 (CHLA-90) slipped into subG1 at 6–24 hours (25–40% specific cell death). The morphological hallmarks of mitotic cell death including defective spindle formation and abnormal cytokinesis were detected by confocal microscopy after the treatment with the vorinostat + flavopiridol combination in CHLA-90. The combination caused reduction in the expression of G2/M proteins (cyclin B1, Mad2, MPM2) in two cell lines with mt TP53, but not in those with wt TP53. Plk1 expression was reduced in all treated lines. siRNA knockdown of Mad2 and cyclin B1 or Plk1 synergistically reduced the clonogenicity of CHLA-90 cells. The combination of HDAC inhibitor and flavopiridol may be a unique approach to treating neuroblastomas with p53-LOF, one that evokes induction of mitotic failure.