Potentiation of camptothecin cytotoxicity by the sequential addition of histone deacetylase inhibitors

This dissertation examines the phenomenon and possible mechanisms of increased cytotoxicity of Camptothecin (CPT) when combined with a Histone Deacetylase inhibitor (HDI). Results describe a defined scheduling protocol showing Histone Deacetylase inhibitors (HDIs) facilitate cytotoxic activity of the Topoisomerase I (Topo I) inhibitor CPT against tumor cells. Cells were treated with CPT and an HDI, either sodium butyrate (NaB) or suberoylanilide hydroxamic acid (SARA). Depending on the sequence of addition, HDI treated cells displayed responses from protection to sensitization, indicating the importance of timing in using HDIs with CPT analogs. Results from the IC50 dose of 15nmol/L CPT when combined with an HDI were similar to the IC80 dose of 100nmol/L CPT alone. Cells arrested in G2/M by CPT were most sensitive to subsequent HDI addition. In CPT arrested cells, NaB decreases Cyclin B levels, as well as levels of anti-apoptotic proteins XIAP and Survivin. Reducing critical anti-apoptotic factors may increase clinical setting Topo I inhibitor efficacy if administered in sequences that do not prevent or inhibit tumor cell progression through S phase.; Dysregulation of the ATM induced G2/M checkpoint and spindle assembly checkpoint (SAC) by HDIs was examined. Cancer cells treated with CPT followed by HDI exhibit decreased levels of ATM pathway activity and important spindle assembly checkpoint proteins. Consequences of signal interference are observed in increased levels of multinucleated cells, a morphological indicator of premature mitosis or mitotic catastrophe. Preliminary data suggests CPT treated cells may have some level of free eukaryotic Initiation Factor 4E (eIF-4E), allowing for the continuation process of cap-dependent translation. In cells treated with CPT + HDI, the phosphorylation pattern of the 4E Binding Protein (Phas1/4EBP1) indicates levels of free eIF-4E may have decreased, leading to decreased cap dependent translation. Results suggest sequential inhibition of Topo I and HDACs can increase tumor cell killing, interfere with two mitotic checkpoints, and can lead to premature mitosis and mitotic catastrophe. The cytotoxic activity of combining these two drugs and their effects on these checkpoints are novel findings, expanding the range of HDI effects in populations of arrested cells. Preliminary results of altered translational regulation and future directions of research are discussed.