Harasym Emily, McAndrew Nicole, Gomez George
Biology Department, University of Scranton, LSC 395, 204 Monroe Ave., 800 Linden Street, Scranton, PA, 18510, USA.
In Vitro Cell Dev Biol Anim. 2017 Oct;53(9):798-809. doi: 10.1007/s11626-017-0190-x. Epub 2017 Aug 24.
Neuroblastoma cells are neural crest derivatives that can differentiate into neuron-like cells in response to exogenous agents, and are known to be particularly sensitive to retinoic acid. The spectrum of neuroblastoma responses, ranging from proliferation, migration, differentiation, or apoptosis, is difficult to predict due to the heterogeneity of these tumors and to the broad effective range of retinoic acid. Our study focused on the effects of nanomolar concentrations of retinoic acid on neuroblastoma differentiation in two cell lines cells: SK-N-SH (HTB-11) and IMR-32. Each cell line was treated with retinoic acid from 1 to 100 nM for up to 6 d. Morphological changes were quantified; immunocytochemistry was used to observe changes in neuronal protein expression and localization, while live-cell calcium imaging utilizing pharmacological agents was conducted to identify neuron-like activity. Retinoic acid-treated HTB-11 but not IMR-32 cells developed specific neuronal phenotypes: acquisition of long neurite-like processes, expression of neurofilament-200, increased responsiveness to acetylcholine, and decreased responsiveness to nicotine and epinephrine. In addition, nanomolar levels of retinoic acid elicited increased nuclear trafficking of the CRABP2, which is traditionally associated with gene expression of cellular pathways related to neuronal differentiation. Collectively, these results show that nanomolar concentrations of retinoic acid are capable of inducing both structural and functional neuron-like features in HTB-11 cells using CRABP2, suggesting differentiation in neuroblastoma cells into neuronal phenotypes. These have important implications for both chemotherapeutic design and for the use of neuroblastomas as in vitro models for neuron differentiation.
神经母细胞瘤细胞是神经嵴衍生物，可对外源因子作出反应分化为神经元样细胞，并且已知对维甲酸特别敏感。由于这些肿瘤的异质性以及维甲酸广泛的有效范围，神经母细胞瘤的反应谱，从增殖、迁移、分化到凋亡，难以预测。我们的研究聚焦于纳摩尔浓度的维甲酸对两种细胞系：SK-N-SH (HTB-11) 和IMR-32 中神经母细胞瘤分化的影响。每个细胞系用1至100 nM的维甲酸处理长达6天。对形态变化进行定量；使用免疫细胞化学观察神经元蛋白表达和定位的变化，同时利用药理学试剂进行活细胞钙成像以识别神经元样活性。经维甲酸处理的HTB-11细胞而非IMR-32细胞形成了特定的神经元表型：获得长的神经突样突起、神经丝-200的表达、对乙酰胆碱反应性增加以及对尼古丁和肾上腺素反应性降低。此外，纳摩尔水平的维甲酸引起CRABP2核转运增加，CRABP2传统上与神经元分化相关的细胞途径的基因表达有关。总体而言，这些结果表明，纳摩尔浓度的维甲酸能够利用CRABP2在HTB-11细胞中诱导结构和功能上的神经元样特征，提示神经母细胞瘤细胞分化为神经元表型。这些对于化疗设计以及将神经母细胞瘤用作神经元分化的体外模型都具有重要意义。
