Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor: effects on gene expression and growth of glioma cells in vitro and in vivo

Abstract

Purpose: Histone acetylation is one of the main mechanisms involved in regulation of gene expression. During carcinogenesis, tumor-suppressor genes can be silenced by aberrant histone deacetylation. This epigenetic modification has become an important target for tumor therapy. The histone deacetylation inhibitor, suberoylanilide hydroxamic acid (SAHA), can induce growth arrest in transformed cells. The aim of this study is to examine the effects of SAHA on gene expression and growth of glioblastoma multiforme (GBM) cells in vitro and in vivo.

Experimental design: The effect of SAHA on growth of GBM cell lines and explants was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Changes of the cell cycle and relative gene expression were detected by fluorescence-activated cell sorting, real-time reverse transcription-PCR, and Western blotting. After glioma cells were implanted in the brains of mice, the ability of SAHA to decrease tumor growth was studied.

Results: Proliferation of GBM cell lines and explants were inhibited in vitro by SAHA (ED50, 2x10(-6) to 2x10(-5) mol/L, 5 days). SAHA exposure of human U87 and T98G glioma cell lines, DA66 and JM94 GBM explants, as well as a murine GL26 GBM cell line resulted in an increased accumulation of cells in G2-M of the cell cycle. Many proapoptotic, antiproliferative genes increased in their expression (DR5, TNFalpha, p21WAF1, p27KIP1), and many antiapoptotic, progrowth genes decreased in their levels (CDK2, CDK4, cyclin D1, cyclin D2) as measured by real-time reverse transcription-PCR and/or Western blot after these GBM cells were cultured with SAHA (2.5x10(-6) mol/L, 1 day). Chromatin immunoprecipitation assay found that acetylation of histone 3 on the p21(WAF1) promoter was markedly increased by SAHA. In vivo murine experiments suggested that SAHA (10 mg/kg, i.v., or 100 mg/kg, i.p.) could cross the blood-brain barrier as shown by prominent increased levels of acetyl-H3 and acetyl-H4 in the brain tissue. Furthermore, the drug significantly (P<0.05) inhibited the proliferation of the GL26 glioma cells growing in the brains of mice and increased their survival.

Conclusions: Taken together, SAHA can slow the growth of GBM in vitro and intracranially in vivo. SAHA may be a welcome addition for the treatment of this devastating disease.