Potentiation of Reactive Oxygen Species Is a Marker for Synergistic Cytotoxicity of MS-275 and 5-Azacytidine in Leukemic Cells.

Epigenetic therapy, the reversal of aberrant epigenetic changes in tumor cells such as the silencing of tumor suppressor genes through means of DNA hypomethylation and histone deacetylation, has become a successful new mode for cancer treatment. Classes of drugs such as histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors are currently being used to target these epigenetic changes. Recently, numerous studies supporting the combination of HDACi and DNA methyltransferase inhibitors have emerged, encouraging early clinical trials of these agents together. MS-275 is a novel and highly active benzamide derivative HDACi that has exhibited in vivo and in vitro anti-proliferative activity toward pancreatic, gastric, lung, and ovarian cancer cells. In addition, MS-275 has been evaluated in Phase I/II clinical trials to treat acute leukemias and solid tumors. 5-azacytidine (Vidaza), a DNA methyltransferase inhibitor, has been shown to reactivate tumor suppressor and DNA repair genes through the hypomethylation of cytosines and is used for treatment of acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). Here we show that MS-275 and 5-azacytidine display synergistic cytotoxicity in AML and ALL (acute lymphocyctic leukemia) cells. Loss of cell viability was increased in a synergistic manner in Jurkat and ML-1 cells treated with the combination of MS-275 and 5-azacytidine. Synergy was assessed using isobologram analysis employed by Calcusyn software. Quantitation of several distinct biochemical apoptotic events, such as DNA fragmentation, phosphotidylserine exposure and caspase-3 activity, also indicated synergistic interactions between MS-275 and 5-azacytidine. Hyperacetylation of histone H3 was also measurably stronger in cells treated with the combination versus either drug alone. Interestingly, intracellular production of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, also increased in a dose dependent and synergistic manner. Since this is a novel marker for this synergism in ALL cells, our data suggest that assessment of oxidative stress can serve as a marker of the concerted action of MS-275 and 5-azacytidine. Recent results from a clinical trial combining MS-275 and 5-azacytidine suggest that the hypomethylation of certain tumor suppressor genes, such as p15, CDH-1, DAPK-1 and SOCS-1, did not correlate with clinical response in myeloid malignancy patients. Thus, identifying new biological markers for clinical activity of these compounds will assist the development of these epigenetic strategies.