Yin Dong, Ong John M, Hu Jinwei, Desmond Julian C, Kawamata Norihiko, Konda Bindu M, Black Keith L, Koeffler H Phillip
Division of Hematology/Oncology and Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, University of California at Los Angeles School of Medicine, Los Angeles, California 90048, USA.
Clin Cancer Res. 2007 Feb 1;13(3):1045-52. doi: 10.1158/1078-0432.CCR-06-1261.
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.
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.
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.
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.
组蛋白乙酰化是参与基因表达调控的主要机制之一。在肿瘤发生过程中，肿瘤抑制基因可因异常的组蛋白去乙酰化而沉默。这种表观遗传修饰已成为肿瘤治疗的重要靶点。组蛋白去乙酰化抑制剂辛二酰苯胺异羟肟酸（SAHA）可诱导转化细胞生长停滞。本研究旨在检测SAHA在体外和体内对多形性胶质母细胞瘤（GBM）细胞基因表达和生长的影响。
采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐检测SAHA对GBM细胞系和外植体生长的影响。通过荧光激活细胞分选、实时逆转录PCR和蛋白质印迹法检测细胞周期变化和相关基因表达。将胶质瘤细胞植入小鼠脑内后，研究SAHA降低肿瘤生长的能力。
SAHA在体外抑制GBM细胞系和外植体的增殖（半数有效剂量，2×10(-6)至2×10(-5)mol/L，5天）。SAHA作用于人U87和T98G胶质瘤细胞系、DA66和JM94 GBM外植体以及小鼠GL26 GBM细胞系后，导致细胞周期G2-M期细胞蓄积增加。实时逆转录PCR和/或蛋白质印迹检测显示，这些GBM细胞用SAHA（2.5×10(-6)mol/L，1天）培养后，许多促凋亡、抗增殖基因表达增加（DR5、TNFα、p21WAF1、p27KIP1），许多抗凋亡、促生长基因水平降低（CDK2、CDK4、细胞周期蛋白D1、细胞周期蛋白D2）。染色质免疫沉淀试验发现，SAHA显著增加了p21(WAF1)启动子上组蛋白3的乙酰化。体内小鼠实验表明，SAHA（10mg/kg，静脉注射，或100mg/kg，腹腔注射）可穿过血脑屏障，脑组织中乙酰化组蛋白H3和乙酰化组蛋白H4水平显著升高即为证明。此外，该药物显著（P<0.05）抑制小鼠脑内GL26胶质瘤细胞的增殖并延长其生存期。
综上所述，SAHA可在体外和体内颅内减缓GBM的生长。SAHA可能是治疗这种毁灭性疾病的一个受欢迎的补充药物。
