Histone deacetylase (HDAC) inhibition modulates intracellular deoxynucleotides (dNTP) pools and potentiates the antitumor effects of the ribonucleotide reductase (RR) inhibitor 3’-Methyl-adenosine (3’-Me-Ado) in promyelocitic leukaemia cell lines.

Abstract

HDAC inhibitors are a new class of antitumor agents that were reported to enhance the cytotoxic effects of a number of classical anticancer drugs through multiple mechanisms. In particular, they are capable of modulating the expression of a series of key cellular genes leading to significant antiproliferative and apoptotic effects. However, which of the possible drug combinations would be the most effective and clinically useful is still to be determined. We treated the HL60 and NB4 promyelocitic leukaemia cells with a combination of the RR inhibitor 3’-Me-Ado and several hydroxamic acid–derived HDAC inhibitors, including the two recently synthesized molecules, MC1864 and MC1879, and the reference compound Trichostatin A (TSA). Initial results showed significant antiproliferative and apoptotic synergistic effects with the combinations. To investigate the molecular basis of this finding, we evaluated the effects of the combinations on cell cycle distribution and on the level of some proteins involved in the apoptotic process (p21, caspase-3, Bcl-2, Bax, AIF). Since HDAC inhibitors seemed to increase the G1-S transition block induced by 3’-Me-Ado, an effect on RR activity was hypothesized. Indeed, the HPLC evaluation of intracellular dNTP pools showed that both TSA and MC1864 induced a significant perturbation in dNTP pools, even if with a somewhat different pattern, suggesting that RR modulation could contribute to the observed synergism. Furthermore, while TSA was shown to activate the intrinsic apoptotic pathway, MC1864 induced a significant dose-dependent increase in ROS generation and the activation of an AIF-dependent apoptotic mechanism. Finally, cell exposure to the radical scavenger N-acetylcysteine determined a significant inhibition of MC1864- but not TSA-mediated synergistic effects. Hence, our findings are consistent with a possible role of HDAC inhibitor mediated-ROS induction in RR inhibition and in the potentiation of RR inhibitors-mediated apoptosis induction.