gms | German Medical Science

Objective: The expression of cellular retinoic acid-binding protein 2 (CRABP2) is significantly lower in short-term survival glioblastoma (GBM) patients (median survival is <12 months) compared with long-term survivals (36 months or longer) and patients with lower grade gliomas. CRABP2 protein is known to directly deliver retinoic acid (RA) to RAR inducing the expression of multiple anti-proliferative genes. SUMOylation is critical for the RA-induced nuclear mobilization of CRABP2 as well as for CRABP2-mediated delivery of RA to RAR. Moreover, epigenetic silencing of this gene has been established as an important process of carcinogenesis. Considering the importance of down-regulation of this protein, our aim was to examine potential regulatory mechanisms of CRABP2 in glioblastoma in vitro.

Method: mRNA and protein expression of CRABP2 from U87MG cell line and 10 GBM tissues were examined using qRT-PCR and Western Blot (WB). In order to detect possible mutation in SUMOylation site, CRABP2 was sequenced. The role of epigenetic silencing on CRABP2 was examined using DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza). MTT assay was used to analyze the cell viability with and without 5-aza treatment and in combination with RA. The role of CRABP2 on cell death was examined by transfecting the cells with pEGFP-CRABP2 plasmid and analyzing the ratio of viable and dead cells by propidium iodide (PI) dye staining.

Results: After amplification and sequencing of CRABP2 coding sequence from U87MG, as well as from 10 GBM tissues, we were unable to detect any mutation in K102, the amino acid responsible for SUMOylation. On the other hand, methylation inhibitor 5-aza up-regulated the expression of CRABP2 in U87MG. Treatment with 5-aza, 5-aza combined with RA, reduces significantly the U87MG cell viability. The best effect was observed by combining 5-aza with RA. U87MG transfected with plasmid coding for CRABP2 showed increased expression of CRABP2 protein and increased cell death by immunofluorescence staining.

Conclusions: 5-aza as a DNA methylation inhibitor can reverse the epigenetic silencing of CRABP2 in U87MG and sensitize glioblastoma cell response to retinoic acid treatment. Over-expressed CRABP2 leads to U87MG cell death in vitro. Since we could not detect any mutation in K102 we assume that SUMOylation, a posttranslational modification responsible for mobilization of the CRABP2 protein to the nucleus and for ist cooperation with RAR, is not accountable for ist down-regulation in the analyzed samples.