Blockade of histone deacetylase inhibitor-induced RelA/p65 acetylation and NF-κB activation potentiates apoptosis in human leukemia cells

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It has been reported that histone deacetylase (HDAC) inhibitors induce NF-κΒ (RelA/p50) activation, and that NF-κB activation status influences the cellular response to HDAC inhibitors. However, relatively little is known about the mechanisms involved in these events. Recently, it has been noted that acetylation of RelA (p65), which is reciprocally regulated by the acetylation mediated by histone acetylases (HATs, e.g., p300 and CBP) and deacetylation mediated by histone deacetylases (e.g., HDAC3). Here we demonstrate that in human leukemia cells, NF-κB activation by the HDAC inhibitors MS-275 and SAHA was associated with hyperacetylation and nuclear translocation of RelA/p65. Co-administration of the IκBα phosphorylation inhibitor Bay 11-7082 or transfection with an IκBα super-repressor increased association of RelA with ΙκΒα, sequestered NF-κB in the cytoplasm, and in so doing, blocked RelA acetylation, which presumably processes in nucleus, and NF-κB activation induced by HDAC inhibitors. Inhibition of NF-κB by pharmacological inhibitors (Bay 11-7082 or SN50) or genetic strategies (transfection with IκBα super-repressor or p65 siRNA) markedly potentiated apoptosis induced by HDAC inhibitors, an action linked to the following NF-κB-associated events. 1) HDAC inhibitors induced ROS generation as well as upregulation of Mn-superoxide dismutase (SOD2), likely a feedback response to increased ROS levels. Bay 11-7082 and IκBα super-repressor enhanced HDAC inhibitor-mediated ROS generation by blocking SOD2 expression, while the SOD mimetic and peroxynitrite scavenger MnTBAP significantly diminished the lethality of Bay 11-7082/HDAC inhibitors. Furthermore, the free radical scavenger N-acetyl L-cysteine (L-NAC) blocked apoptosis induced by Bay 11-7082/HDAC inhibitors by abrogating ROS generation and preventing NF-κB inactivation. 2) Bay 11-7082 and IκBα super-repressor promoted HDAC inhibitor-mediated JNK1 activation. Inhibition of JNK1 activation by either co-treatment with SP600125 or transfection with JNK1 siRNA as well as TAM67 (a transactivation domain-deleted mutation form of c-Jun) attenuated lethality but not ROS production mediated by of Bay 11-7082/HDAC inhibitors. 3) HDAC inhibitors resulted in XIAP down-regulation only in cells co-treated with Bay 11-7082 or transfected with IκBα super-repressor, while XIAP overexpression dramatically protected cells against the Bay 11-7082/HDACI regimen without affecting increased ROS levels. Together, these data suggest that HDAC inhibitors promote accumulation of acetylated RelA in the nucleus, leading to NF-κB activation. Moreover, interference with these events leads to a dramatic increase in HDAC inhibitor-mediated lethality through enhanced oxidative damage, down-regulation of NF-κB-dependent antiapoptotic proteins, and stress-related JNK1 activation.