Schwermer Melanie, Dreesmann Sabine, Eggert Angelika, Althoff Kristina, Steenpass Laura, Schramm Alexander, Schulte Johannes H, Temming Petra
Department of Pediatric Oncology and Hematology, University Hospital Essen, Essen, Germany.
Department of Pediatric Oncology and Hematology, Charité University Medicine Berlin, Berlin, Germany.
Clin Exp Ophthalmol. 2017 Apr;45(3):288-296. doi: 10.1111/ceo.12838. Epub 2016 Nov 10.
Retinoblastoma is the most common malignant cancer of the eye in children. Although metastatic retinoblastoma is rare, cure rates for this advanced disease remain below 50%. High-level polo-like kinase 1 expression in retinoblastomas has previously been shown to be correlated with adverse outcome parameters. Polo-like kinase 1 is a serine/threonine kinase involved in cell cycle regulation at the G2/M transition. Polo-like kinase 1 inhibition has been demonstrated to have anti-tumour effects in preclinical models of several paediatric tumours. Here, we assessed its efficacy against retinoblastoma cell lines.
Expression of polo-like kinase 1 was determined in a panel of retinoblastoma cell lines by polymerase chain reaction and western blot analysis. We analysed viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT assay), proliferation (5-bromo-2'-deoxyuridine enzyme-linked immunosorbent assay), cell cycle progression (propidium iodid staining) and apoptosis (cell death enzyme-linked immunosorbent assay) in three retinoblastoma cell lines after treatment with two adenosine triphosphate-competitive polo-like kinase 1 inhibitors, BI6727 or GSK461364. Activation of polo-like kinase 1 downstream signalling components including TP53 were assessed.
Treatment of retinoblastoma cells with either BI6727 or GSK461364 reduced cell viability and proliferative capacity and induced both cell cycle arrest and apoptosis. Polo-like kinase 1 inhibition also induced the p53 signalling pathway. Analysis of key players in cell cycle control revealed that low nanomolar concentrations of either polo-like kinase 1 inhibitor upregulated cyclin B1 and increased activated cyclin-dependent kinase 1 (phosphorylated at Y15) in retinoblastoma cell lines.
These preclinical data indicate that polo-like kinase 1 inhibitors could be useful as components in rationally designed chemotherapy protocols to treat patients with metastasized retinoblastoma in early phase clinical trials.
视网膜母细胞瘤是儿童最常见的眼部恶性肿瘤。尽管转移性视网膜母细胞瘤很少见，但这种晚期疾病的治愈率仍低于50%。先前已表明视网膜母细胞瘤中高水平的polo样激酶1表达与不良预后参数相关。Polo样激酶1是一种丝氨酸/苏氨酸激酶，参与细胞周期在G2/M期转换的调控。在几种儿科肿瘤的临床前模型中，已证明抑制Polo样激酶1具有抗肿瘤作用。在此，我们评估了其对视网膜母细胞瘤细胞系的疗效。
通过聚合酶链反应和蛋白质印迹分析，在一组视网膜母细胞瘤细胞系中测定polo样激酶1的表达。在用两种三磷酸腺苷竞争性Polo样激酶1抑制剂BI6727或GSK461364处理后，我们分析了三种视网膜母细胞瘤细胞系的活力（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）（MTT法）、增殖（5-溴-2'-脱氧尿苷酶联免疫吸附测定）、细胞周期进程（碘化丙啶染色）和凋亡（细胞死亡酶联免疫吸附测定）。评估了包括TP53在内的Polo样激酶1下游信号成分的激活情况。
用BI6727或GSK461364处理视网膜母细胞瘤细胞可降低细胞活力和增殖能力，并诱导细胞周期停滞和凋亡。抑制Polo样激酶1还可诱导p53信号通路。对细胞周期调控关键因子的分析表明，低纳摩尔浓度的任何一种Polo样激酶1抑制剂均可上调视网膜母细胞瘤细胞系中的细胞周期蛋白B1，并增加活化的细胞周期蛋白依赖性激酶1（Y15位点磷酸化）。
这些临床前数据表明，在早期临床试验中，Polo样激酶1抑制剂可作为合理设计的化疗方案的组成部分，用于治疗转移性视网膜母细胞瘤患者。
