Diesel Britta, Radermacher Jens, Bureik Matthias, Bernhardt Rita, Seifert Markus, Reichrath Jörg, Fischer Ulrike, Meese Eckart
Institut für Humangenetik, Theoretische Medizin, Universität des Saarlandes, Saarbrücken, Germany.
Clin Cancer Res. 2005 Aug 1;11(15):5370-80. doi: 10.1158/1078-0432.CCR-04-1968.
A better understanding of the vitamin D(3) metabolism is required to evaluate its potential therapeutic value for cancers. Here, we set out to contribute to the understanding of vitamin D(3) metabolism in glioblastoma multiforme.
We did nested touchdown reverse transcription-PCR (RT-PCR) to identify CYP27B1 splice variants and real-time RT-PCR to quantify the expression of CYP27B1. A cell line was treated with calcitriol to determine the effect on the expression of CYP27B1, 1alpha,25-dihydroxyvitamin D(3)-24-hydroxylase (CYP24), and vitamin D(3) receptor (VDR). We generated three antibodies for the specific detection of CYP27B1 and splice variants. High-performance TLC was done to determine the endogenous CYP27B1 activity and the functionality of CYP27B1 splice variants. Using WST-1 assay, we determined the effect of vitamin D(3) metabolites on proliferation.
We report a total of 16 splice variants of CYP27B1 in glioblastoma multiforme and a different expression of CYP27B1 and variants between glioblastoma multiforme and normal tissues. We found preliminary evidence for enzymatic activity of endogenous CYP27B1 in glioblastoma multiforme cell cultures but not for the functionality of the splice variants. By adding calcitriol, we found a proliferative effect for some cell lines depending on the dose of calcitriol. The administration of calcitriol led to an elevated expression of CYP27B1 and CYP24 but left the expression of the VDR unaltered.
Our findings show that glioblastoma multiforme cell lines metabolize calcidiol. In addition, we show various effects mediated by calcitriol. We found a special vitamin D(3) metabolism and mode of action in glioblastoma multiforme that has to be taken into account in future vitamin D(3)-related therapies.
为评估维生素D(3)对癌症的潜在治疗价值，需要更好地了解其代谢过程。在此，我们着手促进对多形性胶质母细胞瘤中维生素D(3)代谢的理解。
我们进行巢式降落逆转录PCR（RT-PCR）以鉴定CYP27B1剪接变体，并进行实时RT-PCR以定量CYP27B1的表达。用骨化三醇处理细胞系以确定其对CYP27B1、1α,25-二羟基维生素D(3)-24-羟化酶（CYP24）和维生素D(3)受体（VDR）表达的影响。我们制备了三种用于特异性检测CYP27B1及其剪接变体的抗体。进行高效薄层层析以确定内源性CYP27B1活性及CYP27B1剪接变体的功能。使用WST-1检测法，我们确定了维生素D(3)代谢产物对增殖的影响。
我们报告在多形性胶质母细胞瘤中总共发现了16种CYP27B1剪接变体，且多形性胶质母细胞瘤与正常组织之间CYP27B1及其变体的表达存在差异。我们在多形性胶质母细胞瘤细胞培养物中发现了内源性CYP27B1酶活性的初步证据，但未发现剪接变体具有功能的证据。通过添加骨化三醇我们发现，某些细胞系的增殖效应取决于骨化三醇的剂量。骨化三醇的给药导致CYP27B1和CYP24表达升高，但VDR的表达未改变。
我们的研究结果表明，多形性胶质母细胞瘤细胞系可代谢骨化二醇。此外，我们展示了骨化三醇介导的多种效应。我们在多形性胶质母细胞瘤中发现了一种特殊的维生素D(3)代谢及作用方式，这在未来与维生素D(3)相关的治疗中必须予以考虑。
