Sequential administration of DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) induces apoptosis with caspase and reactive oxygen species (ROS)-dependent synergy

1036 Sequential treatment with DNMTi and HDACi synergize to promote re-expression of transcriptionally silenced genes whose promoters are methylated. This observation has served as a model for several early phase clinical trials in patients with myeloid malignancies and other cancers. Since mechanisms other than gene re-expression may be involved in mediating the clinical efficacy of these drugs, we investigated the effect of sequential treatment of DNMTi and HDACi on apoptosis induction in leukemia cells. Pretreatment of ML-1 or HL-60 with decitabine (DNMTi) for 48 h followed by a variety of HDACi (MS-275, trichostatin A, phenylbutyrate sodium or valproic acid) for 24 h synergistically induced apoptosis (median-effect analysis). Sequential treatment with decitabine followed by HDACi synergistically induced p21 WAF1 expression in ML-1 but not HL-60 cells, indicating that p21 WAF1 upregulation is not essential for apoptotic synergy. ROS can induce apoptosis in caspase-dependent and independent manners. Sequential administration of decitabine and HDACi led to synergistic generation of ROS. Inhibition of ROS by N-acetyl-L-cysteine (NAC) completely abrogated apoptosis. The pan-caspase inhibitor Z-VAD-FMK also completely inhibited apoptosis after sequential administration of decitabine and HDACi. These data suggest that ROS-induced apoptosis requires caspase activation. While sequential administration of DNMTi and HDACi synergize for both re-expression of methylated genes and apoptosis, a possible relationship between these two molecular synergies requires further investigation.