Suberoylanilide Hydroxamic Acid, a Histone Deacetylase Inhibitor: Effects on Gene Expression and Growth of Glioma Cells In vitro and In vivo DongYin, 1 John M. Ong, 2 Jinwei Hu, 2 Julian C. Desmond, 1 Norihiko Kawamata, 1 Bindu M. Konda, 2 Keith L. Black, 2 and H. Phillip Koeffler 1 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 invitro and invivo. 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 cycleandrelative geneexpressionwere detectedby fluorescence-activatedcellsorting,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 invitro by SAHA (ED 50 , 2 Â 10 À6 to 2 Â 10 À5 mol/L, 5 days). SAHA exposure of human U87 andT98G 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 G 2 -M of the cell cycle. Many proapoptotic, antiproliferative genes increased in their expression ( DR5,TNFa ,p21 WAF1 ,p27 KIP1 ), and many antiapoptotic, progrowth genes decreased in their levels ( CDK2,CDK4,cyclinD1,cyclinD2) as measured by real-time reverse transcription-PCR and/or Western blot after these GBM cells were cultured with SAHA (2.5 Â 10 À6 mol/L, 1day). Chromatin immunoprecipitation assay found that acetylation of histone 3 on the p21 WAF1 promoter was markedly increasedby SAHA. Invivo murineexperiments suggested that SAHA (10 mg/kg, i.v., or 100 mg/kg, i.p.) could cross the blood-brain barrier as shownbyprominentincreasedlevelsofacetyl-H3andacetyl-H4inthebraintissue.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 invitro and intracranially invivo. SAHA may be a welcome addition for the treatment of this devastating disease. Histone acetylation is an important regulator of gene expression in higher eukaryotes. It is essential for the normal development of mammals and plays an important physio- logic role (1, 2). Histone deacetylation is mediated by his- tone deacetylases (HDAC). HDACs catalyze the removal of acetyl groups, leading to chromatin condensation and tran- scriptional repression (3, 4). Histone acetyl transferases are enzymes involved in transferring acetyl groups to amino- terminal lysine residues in histones, which results in local expansion of chromatin and increases accessibility of regu- latory proteins to DNA (5). These opposing activities be- tween HDACs and histone acetyl transferases regulate gene expression through chromatin modification (6). In addi- tion, methylated DNA-binding proteins can bind specific- ally to methylated DNA to cause transcriptional silencing (7), and once methylated DNA-binding proteins bind to DNA, HDACs are recruited by a separate transcriptional repressor domain (8–10), resulting in long-term silencing of the target gene (11, 12). Cancer Therapy: Preclinical Authors’Affiliations: 1 Division of Hematology/Oncology and 2 Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, University of California at Los Angeles School of Medicine, Los Angeles, California Received5/24/06;revised10/20/06;accepted11/9/06. Grant support: NIH,Tom Collier Foundation, Parker HughesTrust, Inger Fund, Maxine Dunitz Neurosurgical Institute, and Cedars-Sinai Medical Center. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: D. Yin and J.M. Ong contributed equally to this work and should be con- sidered co ^ first authors. K.L. Black and H.P. Koeffler contributed equally to this work and should be considered co-last authors. H.P. Koeffler is a member of the Molecular Biology Institute andJonsson Comprehensive CancerCenterat University ofCaliforniaatLosAngelesandholdstheendowedMarkGoodsonChairofOncology Research at Cedars-Sinai Medical Center/University of California at Los Angeles SchoolofMedicine.ThisworkisinlovingmemoryofMattSchreck. Requestsforreprints: DongYin, Division of Hematology and Oncology, Cedars- Sinai Medical Center, University of California at Los Angeles School of Medicine, 8700 Beverly Boulevard, Davis Building, Room 5022, Los Angeles, CA 90048. Phone: 310-423-7740; Fax: 310-423-0225; E-mail: Dong.Yin@cshs.org. F 2007 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-1261 www.aacrjournals.org ClinCancerRes2007;13(3)February1,2007 1045 Research. on April 10, 2017. © 2007 American Association for Cancer clincancerres.aacrjournals.org Downloaded from