Antitumor effects of novel co-drugs linking histone deacetylase and ribonucleotide reductase inhibitors in hematological tumors

Abstract

Combination therapy is the mainstay of anticancer therapy due to the significant synergistic effects achievable. Now that anticancer drug research turned toward a more molecular targeted approach, the design of dual-target drugs appears to be a new promising strategy with the potential to improve the therapeutic efficacy of the single drug and to reduce the probability of drug induced resistance and cross resistance. In our previous work, we found that 3’-C-methyl-adenosine (3’-Me-Ado), developed by us as a potent ribonucleotide reductase (RR) inhibitor with antitumor activity against both human leukemia and carcinoma cell lines, elicited significant growth inhibitory and apoptotic synergistic effects in promyelocytic leukemia cells in combination with some hydroxamic acid–derived histone deacetylase (HDAC) inhibitors. Also the antiepileptic agent valproic acid (VPA) was shown to inhibit HDAC in tumors and to synergize with a number of anticancer drugs including RR inhibitors. Since VPA is currently used and well tolerated in the clinics, we pursued the development of co-drugs by combining the structures of 3’-Me-Ado and VPA in a single molecule. Thus, the 3’-C-methyladenosine-2’(5’)-O-valproic esters (A157, A167) and the 2’,5’-diester analog (A160) were synthesized and evaluated for their antitumor potential in HL60 and HuT78 cells. Cytotoxicity was studied by the Trypan blue dye exclusion test, whereas apoptosis was evaluated by fluorescence microscopy after staining with acridine orange and etidium bromide. Preliminary results showed that the compounds induced cytotoxic effects with IC50 ranging from 11 and 600 μM and from 32 and 900 μM in HL60 and HuT78 respectively, demonstrating a higher potency when compared with VPA alone (IC50 of about 2.5 mM). However, their apoptotic activity was modest, with the most potent compound A160 showing an AC50 higher than 100 μM in HL60 cells. The western blotting study of the level of acetylated histone H3 demonstrated that the compounds were not capable of inhibiting significantly the HDAC enzymes. Instead, the evaluation of cell cycle distribution by flow cytometry did show a recruitment of treated cells in the G1 phase of the cell cycle, suggesting that the compounds could retain the RR inhibiting activity of 3’-Me-Ado. Further studies are in progress to clarify the mechanism of cytotoxic activity of the synthesized co-drugs.