Notch- and vitamin D signaling in 1,25(OH)2D3-resistant glioblastoma multiforme (GBM) cell lines

Abstract

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.