HDAC inhibitor valproic acid increases CRABP2 expression and in combination with retinoic acid synergistically inhibits proliferation in glioblastoma cells

HDAC inhibitor valproic acid increases CRABP2 expression and in combination with retinoic acid synergistically inhibits proliferation in glioblastoma cells

Lei Yao

Doctoral thesis

Date of Examination: 2016-12-14

Date of issue: 2016-12-12

Advisor: Rohde, Veit Prof. Dr.

Referee: Dobbelstein, Matthias Prof. Dr.

Referee: Thelen, Paul PD Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-002B-7CD0-F

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Abstract

English

Background: Although integrated treatments were implemented, glioblastoma multiforme (GBM) remains one of the most lethal primary brain tumors. The cellular retinoic acid–binding protein 2 (CRABP2) plays a crucial role, as one of the key cytoplasmic transport-proteins in retinoic acid (RA) pathway in various cancers, hence in GBM. Downregulation of CRABP2 protein and its lower ratio to fatty acid–binding protein 5 (FABP5) were showed to play important role in impaired RA pathway and in survival time of GBM patients. The specific mechanism of CRABP2 regulation in GBM is still not clear. Methods: Luciferase Reporter Assay was used to test hsa-mir-34a and hsa-mir-1262 mediated effects on 3’- untranslated region (3’UTR) of CRABP2 mRNA. In order to detect possible mutation in SUMOylation site, coding sequence of CRABP2 from different GBM tissues and cell lines was amplified and sequenced. Valproic acid (VPA) as histone deacetylation inhibitor, 5-aza-2'-deoxycytidine (5-aza) as DNA methylation Inhibitor and RA were used to treat U87MG and patient-derived 36st GBM cell lines. Real-time PCR was used for mRNA quantification of CRABP2, FABP5, methyltransferase 1 (DNMT1), Caspase 7 and Bcl-2 DNA in cells treated with VPA, VPA+RA, 5-aza as well as in cells transfected with pcDNA4-CRABP2 plasmid. MTT assays were performed to detect GBM cells viability after VPA single and 5-aza single and in combination with RA treatment. Immunocytochemistry (ICC) was performed to detect expression of CRABP2 using anti-CRABP2, and proliferation level using anti-Ki-67 antibody. Results: Downregulation of CRABP2 in GBM is not due to its modulation by hsa-mir-34a and hsa-mir-1262 or a mutation in Lysine 102. VPA treatment alone and in combination with RA increased CRABP2 expression and inhibited proliferation and cell viability in GBM cells. Moreover, VPA treatment as well as 5-aza decreased DNMT 1 level in vitro. Besides that, both over-expression of CRABP2 as well as VPA treatment, increased apoptosis in glioblastoma cells. Conclusions: VPA increases expression of CRABP2 in analyzed GBM cell lines. Additional increase of CRABP2 expression was achieved by combined VPA and RA treatment. VPA+RA drug combination synergistically inhibited the proliferation of glioblastoma cells. This data suggests that VPA in combination with RA can be a promising anti-cancer therapy for GBM.

Keywords: Glioblastoma; CRABP2; valproic acid; retinoic acid