Histone Deacetylase Inhibitor RGFP109 Overcomes Temozolomide Resistance by Blocking NF-κB-Dependent Transcription in Glioblastoma Cell Lines

• Published in: Neurochemical research 2016-12, Vol.41 (12), p.3192-3205
• Main Authors: Li, Zong-yang, Li, Qing-zhong, Chen, Lei, Chen, Bao-dong, Wang, Bo, Zhang, Xie-jun, Li, Wei-ping
• Format: Article
• Language: English
• Subjects: Acetylation; Active Transport, Cell Nucleus; Antineoplastic Agents, Alkylating - pharmacology; Apoptosis; Benzamides - pharmacology; Binding; Biochemistry; Biomedical and Life Sciences; Biomedicine; Blocking; Cell Biology; Cell Cycle Proteins - metabolism; Cell Line, Tumor; Cell Nucleus - metabolism; Cell survival; Central nervous system; Central Nervous System Neoplasms; Dacarbazine - analogs & derivatives; Dacarbazine - pharmacology; Down-regulation; Drug Resistance, Neoplasm - drug effects; Drug Synergism; Electrophoretic mobility; Genes; Glioblastoma; Glioma; Histone deacetylase; Histone Deacetylase Inhibitors - pharmacology; Histones; Homeodomain Proteins - metabolism; Humans; Immunofluorescence; Immunoprecipitation; Inhibitors; Neurochemistry; Neurology; Neurosciences; NF-kappa B - genetics; NF-kappa B - metabolism; NF-κB protein; Nuclear transport; Original Paper; Phosphorylation; Plasmids; Post-translation; Signal Transduction; Temozolomide; Transcription; Transcription Factor RelA - genetics; Transcription Factor RelA - metabolism; Transcription, Genetic; Transcriptional Activation; Translocation; Tumor cell lines; Tumor Suppressor Proteins - metabolism
• ISSN: 0364-3190; 1573-6903; 1573-6903
• DOI: 10.1007/s11064-016-2043-5

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.