Department of Internal Medicine I, The Saarland University Hospital, Homburg/Saar, Germany.
J Steroid Biochem Mol Biol. 2010 Jul;121(1-2):420-4. doi: 10.1016/j.jsbmb.2010.02.028. Epub 2010 Mar 4.
Recently, an important role of Notch activation for Ras-induced transformation of glial cells and for glioma growth and survival has been demonstrated. It was concluded that activation of Notch-signaling may represent a new target for glioblastoma multiforme (GBM) therapy. We now analyzed five GBM cell lines (Tx3095, Tx3868, U87, U118, U373) for key components of Notch-signaling pathways (Notch-1, Notch-2, Notch-3, Notch-4, Delta-like 1, Delta-like 3, Delta-like 4, Jagged-1, Jagged-2) using conventional RT-PCR. We found that some components (Notch-1, Notch-2, Notch-4, Jagged-1) were consistently expressed in all cell lines analyzed while, in contrast, other key components of Notch-signaling were differentially expressed. Notch-3 was expressed in three out of five cell lines (in U87, U118 and U373), but was missing in Tx3095 and Tx3868 cells. Jagged-2 was expressed in U87, U373 and Tx3868, but not in U118 or Tx3095 cells. Delta-like 1 and Delta-like 3 were not detected in Tx3905 cells, but in all other cell lines. RNA for Delta-like 4 was only found in U373 and Tx3868 GBM cell lines. Treating GBM cell lines with 1,25(OH)2D3 (10(-6), 10(-8), and 10(-10) M), the biologically active form of vitamin D, did not result in significant dose- or time-dependent antiproliferative effects, indicating that GBM cell lines are resistant against the antiproliferative activity of 1,25(OH)2D3. In vitro treatment of GBM cells with 1,25(OH)2D3 did not result in a modulation of the expression of key components of the Notch-signaling pathway. Treatment with HDAC-inhibitor TSA or DNA-methyltransferase inhibitor 5-aza exerted dose- and time-dependent antiproliferative effects on GBM cell lines. We asked the question whether the resistance against 1,25(OH)2D3 could be restored by co-treatment with TSA or 5-aza. However, combination therapy with 1,25(OH)2D3 and TSA or 5-aza did not result in enhanced antiproliferative effects as compared to treatment with TSA or 5-aza alone. In contrast, antiproliferative effects of TSA and 5-aza were partially antagonized by concomitant treatment with 1,25(OH)2D3, indicating a protective effect of 1,25(OH)2D3 against the antiproliferative effects of TSA and 5-aza in GBM cell lines. In conclusion, our findings point at a differential expression of key components of Notch-signaling in GBM cell lines that may be of importance for the growth characteristics of GBM. Our findings indicate that GBM cell lines are resistant against the antiproliferative effects of 1,25(OH)2D3, and that this resistance may not be overcome by modulation of epigenetic silencing. Our findings do not support the hypothesis that modulation of Notch-signaling pathways by 1,25(OH)2D3 may regulate growth of GBM cell lines.
最近，Notch 激活在 Ras 诱导的神经胶质细胞转化和神经胶质瘤生长和存活中的重要作用已经得到证明。研究结论表明，Notch 信号通路的激活可能成为多形性胶质母细胞瘤（GBM）治疗的新靶点。我们现在使用常规 RT-PCR 分析了 5 种 GBM 细胞系（Tx3095、Tx3868、U87、U118、U373）中 Notch 信号通路的关键组成部分（Notch-1、 Notch-2、 Notch-3、 Notch-4、Delta-like 1、Delta-like 3、Delta-like 4、Jagged-1、Jagged-2）。我们发现，一些成分（Notch-1、 Notch-2、 Notch-4、Jagged-1）在所有分析的细胞系中均持续表达，而其他关键的 Notch 信号成分则表现出不同的表达水平。Notch-3 在 5 种细胞系中的 3 种（U87、U118 和 U373）中表达，但在 Tx3095 和 Tx3868 细胞中缺失。Jagged-2 在 U87、U373 和 Tx3868 中表达，但不在 U118 或 Tx3095 细胞中表达。Tx3905 细胞中未检测到 Delta-like 1 和 Delta-like 3，但在所有其他细胞系中均有检测到。仅在 U373 和 Tx3868 GBM 细胞系中检测到 Delta-like 4 的 RNA。用 1,25(OH)2D3（10^(-6)、10^(-8)和 10^(-10) M）处理 GBM 细胞系，1,25(OH)2D3 是维生素 D 的生物活性形式，未导致明显的剂量或时间依赖性抗增殖作用，表明 GBM 细胞系对 1,25(OH)2D3 的抗增殖活性具有抗性。体外用 1,25(OH)2D3 处理 GBM 细胞不会导致 Notch 信号通路关键成分的表达发生变化。用 HDAC 抑制剂 TSA 或 DNA 甲基转移酶抑制剂 5-aza 处理 GBM 细胞系，会导致剂量和时间依赖性的抗增殖作用。我们提出了一个问题，即 1,25(OH)2D3 的耐药性是否可以通过 TSA 或 5-aza 的联合治疗来恢复。然而，与 TSA 或 5-aza 单独治疗相比，1,25(OH)2D3 与 TSA 或 5-aza 的联合治疗并没有增强抗增殖作用。相反，TSA 和 5-aza 的抗增殖作用部分被同时用 1,25(OH)2D3 治疗所拮抗，表明 1,25(OH)2D3 对 TSA 和 5-aza 在 GBM 细胞系中的抗增殖作用具有保护作用。总之，我们的研究结果表明，GBM 细胞系中 Notch 信号通路的关键成分表达存在差异，这可能对 GBM 的生长特征很重要。我们的研究结果表明，GBM 细胞系对 1,25(OH)2D3 的抗增殖作用具有抗性，这种抗性不能通过表观遗传沉默的调节来克服。我们的研究结果并不支持 Notch 信号通路的调节可以通过 1,25(OH)2D3 来调节 GBM 细胞系生长的假设。
