A Novel Histone Deacetylase Inhibitor MCT-3 Augments Inhibition of Leukemia Cell Growth and Suppresses Induction of MMP-9 Expression by Azacitidine in HL-60 Cells.

Azacitidine (AZA), a demethylating agent, has recently been demonstrated to have efficacy in the treatment of myelodysplasia and acute myeloid leukemia. A potential concern when considering the use of this agent is a recent report demonstrating AZA-mediated re-activation of matrix metalloproteinase-9 (MMP-9) expression facilitating the invasive metastatic phenotype (Sato NM et al J Natl Cancer Inst. 2003 Feb 19;95(4):327–30). Histone deacetylase inhibitors (HDACi) are a recently identified class of agents with considerable in vitro and in vivo activity and early phase clinical efficacy in the treatment of haematological malignancies (Bolden JE Nat Rev Drug Discov. 2006 Sep;5(9):769–84). Pre-clinical in vitro studies suggest that addition of HDACi to 5-aza-2′-deoxycytidine, a derivative of AZA, enhances the efficacy of this agent (Yang H et al Leuk Res. 2005 Jul; 29(7):739–48) whilst early phase clinical trials identify therapeutic activity using a combination of demethylating agents and HDACi (Garcia-Manero G, Blood. 2006 Nov 15;108(10):3271–9). Our current study aimed to determine the in vitro activity and molecular mechanisms of action of the novel HDACi MCT-3, a derivative of Oxamflatin, a hydroxamate analogue, (Dear AE, et al, Org Biomol Chem, 2006, 4, 3778–3784) in the HL-60 cell line alone and combination with AZA. AZA (1.0 microM) and MCT-3 (2.5 microM) alone inhibited HL-60 cell growth over 24hrs by 40%, 30% respectively. The combination of AZA with MCT-3 inhibited HL-60 cell growth up to 50%. Real-time PCR demonstrated that AZA and MCT-3 alone increased p15^INK4b and Caspase 3 mRNA expression 2 fold. A Combination of AZA with MCT-3 increased p15^INK4b and Caspase 3 mRNA expression up to 2.5 and increased p21^WAF1/CIP1 and the orphan nuclear receptor Nur77 expression 2 fold. A combination of AZA and MCT-3 significantly attenuated AZA-induced MMP-9 mRNA expression and proteolytic activity. AZA and MCT-3 alone reduce HL-60 cell growth in vitro. Addition of MCT-3 to AZA increased inhibition of cell growth, suggesting that this HDACi may have the potential for additive activity with demethylating agents. AZA and MCT-3 have similar effects on expression of genes implicated in cell cycle arrest and apoptosis. Increased expression of p21^WAF1/CIP1 and the orphan nuclear receptor Nur77 via inhibition of cell cycle progression and enhanced apoptosis may in part be responsible for the enhanced anti-leukaemia activity of the combination of AZA and MCT-3. Importantly MCT-3 is able to inhibit AZA-mediated induction of MMP-9 expression.