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

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.

Reichrath S ，Müller CS ，Gleissner B ，Pfreundschuh M ，Vogt T ，Reichrath J ... -  《-》

VDR microRNA expression and epigenetic silencing of vitamin D signaling in melanoma cells.

Malignant melanoma cells express the vitamin D receptor (VDR). However, some melanoma cell lines fail to respond to the antiproliferative effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We reported previously that out of seven melanoma cell lines analyzed, three cell lines (MeWo, SK-Mel28, SM) respond to the antiproliferative effects of 1,25(OH)2D3, while the others (SK-Mel5, SK-Mel25, IGR, Meljuso) are resistant. It was the aim of this study to investigate whether epigenetic mechanisms are of importance for the abrogation of vitamin D signaling in vitamin D resistant melanoma cells. We used the histone deacetylase inhibitor (HDACI) trichostatin A (TSA) and the DNA methyltransferase inhibitor (DNMTI) 5-azacytidine (5-Aza) to elucidate the effects of protein acetylation and of DNA hypermethylation on 1,25(OH)2D3-induced effects on cell proliferation, respectively. Additionally we analyzed the expression of VDR microRNA in 1,25(OH)2D3-responding and resistant melanoma cells. TSA and 5-Aza exerted dose- and time-dependent antiproliferative effects on melanoma cell lines. Interestingly, combination therapy with 1,25(OH)2D3 and TSA exerted synergistic antiproliferative effects in a 1,25(OH)2D3-resistant melanoma cell line (IGR) (p<0.05). Combination therapy with 1,25(OH)2D3 and 5-Aza resulted in synergistic (MeWo after 72 h; p<0.05) or additive (other melanoma cell lines analyzed) antiproliferative effects. Additionally, we could show that VDR mRNA expression is relatively high in two of three 1,25(OH)2D3-responsive melanoma cells as compared to resistant cells, moreover this relatively high VDR expression is associated with low expression of miRNA125b in MeWo and SK-Mel28 cells. Our results suggest that the endogenous VDR mRNA level is inversely associated with expression of miRNA125b in melanoma cell lines analyzed. Moreover, miRNA125b may be involved in the regulation of VDR expression and in the resistance against 1,25(OH)(2)D(3) in melanoma cells. It can be speculated whether miRNA125b may be of prognostic importance and/or may represent a therapeutic target for malignant melanoma. Drugs that influence epigenetic mechanisms might be promising therapeutics for the treatment of metastasized malignant melanoma, alone or in combination with antiproliferative or cytotoxic agents such as 1,25(OH)2D3.

Essa S ，Denzer N ，Mahlknecht U ，Klein R ，Collnot EM ，Tilgen W ，Reichrath J ... -  《-》

Contribution of Notch signaling activation to human glioblastoma multiforme.

Kanamori M ，Kawaguchi T ，Nigro JM ，Feuerstein BG ，Berger MS ，Miele L ，Pieper RO ... -  《JOURNAL OF NEUROSURGERY》

Specific knockdown of uPA/uPAR attenuates invasion in glioblastoma cells and xenografts by inhibition of cleavage and trafficking of Notch -1 receptor.

Raghu H ，Gondi CS ，Dinh DH ，Gujrati M ，Rao JS ... -  《Molecular Cancer》

Antiproliferative signalling by 1,25(OH)2D3 in prostate and breast cancer is suppressed by a mechanism involving histone deacetylation.

Banwell CM ，Singh R ，Stewart PM ，Uskokovic MR ，Campbell MJ ... -  《-》