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

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.

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S1237 Temozolomide Temozolomide (CCRG81045, NSC 362856, TMZ) is a monofunctional SN-1 alkylating agent that can modify nitrogen atoms in the DNA ring and the extracyclic oxygen group, chemically converted to MTIC and degrades to methyldiazonium cation, which transfers methyl groups to DNA at physiologic pH. A DNA damage inducer in L-1210 and L-1210/BCNU cells. Temozolomide induces apoptosis and exhibits antitumor activity. (106) (7)

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