HDAC抑制剂LBH589对人多形性胶质母细胞瘤增殖和浸润的作用机制研究

Glioblastoma multiforme (GBM) is the one of malignant primary brain tumor in humans with strong proliferative capacity, high invasiveness and poor prognosis. GBM patients obtain a significant survival benefit from adjuvant chemotherapy. Previous studies have found various histone deacetylases (HDACs) inhibitors generate clinically significant anti-GBM effects. However, the relationship between their inhibition of HDACs and their anti-GBM effects is still unknown. Our previous study found that, the GBM cell line treated by HDAC inhibitor LBH589 performed significantly increased histone acetylation and p21WAF1 expression, and inhibited HIF-1α expression. Use chromatin immunoprecipitation, Western blotting, small animal imaging (MRI), and other methods in this study, we intend to further clarify effects of LBH589 on ① GBM proliferation and invasion, ② nuclear HDACs located in the promoter region of p21WAF1 gene, ③ nuclear and cytoplasmic HDACs to regulate HIF-1α stability and angiogenesis factors expression. We hope to provide insights for new drug targets and experimental basis for improving the efficacy of chemotherapy for GBM.

多形性胶质母细胞瘤（GBM）具有增殖能力强、侵袭力高、预后差等特点。术后辅助化疗对延长GBM患者生存时间具有重要意义。目前多种组蛋白去乙酰化酶（HDACs）抑制剂均有抗GBM作用，但其抑制HDACs活性的作用与其抗GBM作用之间的联系未见报道。我们前期研究发现，HDAC抑制剂LBH589可增加GBM细胞株组蛋白乙酰化和增殖抑制蛋白p21WAF1水平并降低浸润相关蛋白HIF-1α水平。本项目拟采用染色质免疫共沉淀、免疫印迹、小动物MRI活体成像等方法在细胞和动物水平进一步阐明LBH589：①是否抑制GBM增殖和浸润？②是否通过抑制基因启动子区HDACs以促进p21WAF1表达？③是否通过抑制胞浆和胞核内HDACs以促进HIF-1α降解并抑制血管生成因子表达？本项目将为提高GBM化疗效果提供新的药物靶点和实验依据。

研究背景及目的.多形性胶质母细胞瘤（GBM）是一种高度血管化的恶性脑肿瘤。GBM内血管的生成对肿瘤生长、侵袭、临床复发和预后均有重要意义。LBH589（又名Panobinostat）是一种定位于细胞浆和细胞核内的非选择性蛋白质去乙酰化酶（HDAC）抑制剂，对多种血液肿瘤和实体肿瘤的生长均有显著抑制作用。本研究拟探讨LBH589对GBM血管生成的作用及其机制。.主要研究内容及方法.U87MG细胞培养于缺氧环境中（1% O2、5% CO2和94% N2），DMSO组为溶剂对照组。应用CCK-8法检测不同作用时间后LBH589对U87MG细胞增殖活性的影响。应用U87MG荷瘤小鼠检测LBH589对移植瘤生长的影响；应用免疫组化方法检测瘤体内微血管密度（MVD）。应用HDAC活性试剂盒和Western blotting方法检测LBH589对U87MG细胞内HDAC活性和蛋白质乙酰化修饰水平的影响；应用Western blotting、RT-PCR方法LBH589对缺氧环境下U87MG细胞内HIF-1α、VEGF mRNA和蛋白质表达的影响；应用免疫共沉淀方法鉴定U87MG细胞内与HIF-1α相互作用的蛋白质。.研究结果及数据.1、LBH589显著抑制U87MG细胞株增殖活性和荷瘤小鼠瘤体的生长.2、LBH589显著抑制荷瘤小鼠瘤体内血管生成.3、LBH589显著抑制U87MG细胞内HDAC活性和蛋白质去乙酰化修饰.4、LBH589抑制HSP90/HDAC6复合物活性和HIF-1α、VEGF的表达.结论及意义.LBH589通过抑制HSP90/HDAC6活性、促进HIF-1α的降解、减少促血管生成因子的表达以抑制GBM的血管生成和细胞增殖。本研究不仅为胶质母细胞瘤的治疗提供了新的思路和作用靶点，也为其他肿瘤的抗血管生成研究了参考。