Abstract
Nerve/glial antigen (NG)2 enhances cell proliferation, migration as well as chemoresistance and is associated with a poor clinical outcome in glioblastoma. Since regulatory mechanisms of NG2 expression are still largely unknown, we herein investigated the impact of protein kinase CK2 inhibition on NG2 expression in human glioblastoma cells. CK2 was inhibited in NG2-positive human glioblastoma cell lines (A1207, U87) and primary human glioblastoma cells by pharmacological treatment (CX-4945, TBB) or siRNA. NG2 expression was analyzed by flow cytometry, Western Blot and qRT-PCR. Cytotoxicity and viability were assessed by WST-1, LDH and BrdU assays. Scratch, sprouting and BrdU assays as well as growth curves were performed to examine cell migration and proliferation. Truncated fragments of the human NG2 promotor were generated and their transcriptional activity was assessed using reporter gene assays. The effect of CK2 inhibition on tumor growth was investigated in xenografts within the flanks of NOD/SCID mice. Finally, effects of CK2 inhibition were analyzed in primary human glioblastoma cells. We found that inhibition of CK2 significantly reduces NG2 protein levels in A1207 (19%±6.2; Mean±SD), U87 (35%±11.3) and primary human glioblastoma cells (41%) when compared to controls. Further analyses revealed that this is due to a decreased NG2 mRNA level. Of note, we identified a 200 base pair fragment, including a binding site for the CK2-dependent transcription factor SP-1. Functional assays showed no cytotoxic effects of the decreased NG2 expression after CK2 inhibition, whereas cell migration and proliferation were markedly reduced. Moreover, we found that CX-4945 treatment decreases tumor growth, which is associated with a diminished NG2 expression (56%±13.0) when compared to controls. In conclusion, we identified CK2 as a novel regulator of NG2 gene expression in human glioblastoma cells. Hence, pharmacological inhibition of CK2 may represent a novel strategy in the therapy of NG2-positive glioblastoma.
Rutaecarpine Inhibits U87 Glioblastoma Cell Migration by Activating the Aryl Hydrocarbon Receptor Signaling Pathway
