Li Zong-Yang, Li Qing-Zhong, Chen Lei, Chen Bao-Dong, Wang Bo, Zhang Xie-Jun, Li Wei-Ping
Brain Center, Department of Neurosurgery, Shenzhen Key Laboratory of Neurosurgery, Shenzhen Second People's Hospital, The Clinical College of Anhui Medical University, Shenzhen University 1st Affiliated Hospital, 3002# Sungang Road, Futian district, Shenzhen, 518035, China.
Shantou University Medical College, 22# Xinling Road, Shantou, Guangdong, China.
Neurochem Res. 2016 Dec;41(12):3192-3205. doi: 10.1007/s11064-016-2043-5. Epub 2016 Sep 8.
Glioblastoma (GBM) is the most frequent and aggressive tumour in the central nervous system. Many studies have demonstrated that upregulation of the NF-κB onco-pathway is accompanied by the acquisition of Temozolomide (TMZ) resistance in GBM cells. Here, we show that RGFP109, a selective histone deacetylase (HDAC1 and HDAC3) inhibitor, overcomes TMZ resistance and downregulates the expression of NF-κB-regulated pro-survival genes in a TMZ-resistant (TR) GBM cell line. RGFP109 did not alter the phosphorylation levels of NF-κB/p65 or inhibitory κBα (IκBα). Immunofluorescence microscopy showed that RGFP109 does not block the nuclear translocation of NF-κB/p65. However, co-immunoprecipitation assays revealed that RGFP109 induces the hyperacetylation of NF-κB/p65 and histones, and blocks interactions between NF-κB/p65 and its coactivators, p300 and p300/CBP-associated factor (PCAF). These results indicate that RGFP109-mediated post-translational nuclear acetylation may be involved in the regulation of NF-κB. Electrophoretic mobility shift assays revealed that RGFP109 reduces NF-κB/p65 binding to κB-DNA and decreased the transcriptional level of κB-mediated genes, suggesting that RGFP109-induced hyperacetylation leads to attenuated transcription of the κB gene. In addition, RGFP109 elevates the expression of inhibitor of growth 4 (ING4), which is typically downregulated in GBM cells. Importantly, we found that RGFP109 enhances ING4 recognition and binding to NF-κB/p65, which may be positively correlated with reduced interactions between NF-κB/p65 and p300/PCAF, thereby effecting transcription of the κB gene. Finally, we show that knockdown of ING4 with plasmids containing pcDNA3.1-ING4 shRNA abolished the effect of RGFP109. Therefore, ING4 may act as a corepressor and facilitate RGFP109-triggered suppression of the NF-κB pathway. Taken together, our data show that RGFP109, an HDAC inhibitor, in combination with TMZ may be a therapeutic candidate for patients with temozolomide-resistant GBM.
胶质母细胞瘤（GBM）是中枢神经系统中最常见且侵袭性最强的肿瘤。许多研究表明，NF-κB致癌通路的上调与GBM细胞中替莫唑胺（TMZ）耐药性的获得有关。在此，我们表明RGFP109，一种选择性组蛋白脱乙酰酶（HDAC1和HDAC3）抑制剂，可克服TMZ耐药性，并下调TMZ耐药（TR）GBM细胞系中NF-κB调节的促生存基因的表达。RGFP109未改变NF-κB/p65或抑制性κBα（IκBα）的磷酸化水平。免疫荧光显微镜检查显示，RGFP109不会阻止NF-κB/p65的核转位。然而，免疫共沉淀试验表明，RGFP109诱导NF-κB/p65和组蛋白的超乙酰化，并阻断NF-κB/p65与其共激活因子p300和p300/CBP相关因子（PCAF）之间的相互作用。这些结果表明，RGFP109介导的翻译后核乙酰化可能参与NF-κB的调节。电泳迁移率变动分析显示，RGFP109减少NF-κB/p65与κB-DNA的结合，并降低κB介导基因的转录水平，表明RGFP109诱导的超乙酰化导致κB基因转录减弱。此外，RGFP109提高生长抑制因子4（ING4）的表达，ING4在GBM细胞中通常下调。重要的是，我们发现RGFP109增强ING4对NF-κB/p65的识别和结合，这可能与NF-κB/p65与p300/PCAF之间相互作用的减少呈正相关，从而影响κB基因的转录。最后，我们表明用含有pcDNA3.1-ING4 shRNA的质粒敲低ING4可消除RGFP109的作用。因此，ING4可能作为一种共抑制因子，并促进RGFP109触发的NF-κB通路抑制。综上所述，我们的数据表明，HDAC抑制剂RGFP109与TMZ联合使用可能是替莫唑胺耐药GBM患者的一种治疗候选方案。
