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The purinergic system is highly involved in the regulation of microglial physiological processes. In addition to the accepted roles for the P2X4,7 and P2Y12 receptors activated by adenosine triphosphate (ATP) and adenosine diphosphate, respectively, recent evidence suggests a role for the adenosine A2A receptor in microglial cytoskeletal rearrangements. However, the expression and function of adenosine A1 receptor (A1AR) in microglia is still unclear. Several reports have demonstrated possible expression of A1AR in microglia, but a new study has refuted such evidence. In this study, we investigated the presence and function of A1AR in microglia using biomolecular techniques, live microscopy, live calcium imaging, and in vivo electrophysiological approaches. The aim of this study was to clarify the expression of A1AR in microglia and to highlight its possible roles. We found that microglia express A1AR and that it is highly upregulated upon ATP treatment. Moreover, we observed that selective stimulation of A1AR inhibits the morphological activation of microglia, possibly by suppressing the Ca2+ influx induced by ATP treatment. Finally, we recorded the spontaneous and evoked activity of spinal nociceptive-specific neuron before and after application of resting or ATP-treated microglia, with or without preincubation with a selective A1AR agonist. We found that the microglial cells, pretreated with the A1AR agonist, exhibit lower capability to facilitate the nociceptive neurons, as compared with the cells treated with ATP alone. The purinergic system is highly involved in the regulation of microglial physiological processes. In addition to the accepted roles for the P2X4,7 and P2Y12 receptors activated by adenosine triphosphate (ATP) and adenosine diphosphate, respectively, recent evidence suggests a role for the adenosine A2A receptor in microglial cytoskeletal rearrangements. However, the expression and function of adenosine A1 receptor (A1AR) in microglia is still unclear. Several reports have demonstrated possible expression of A1AR in microglia, but a new study has refuted such evidence. In this study, we investigated the presence and function of A1AR in microglia using biomolecular techniques, live microscopy, live calcium imaging, and in vivo electrophysiological approaches. The aim of this study was to clarify the expression of A1AR in microglia and to highlight its possible roles. We found that microglia express A1AR and that it is highly upregulated upon ATP treatment. Moreover, we observed that selective stimulation of A1AR inhibits the morphological activation of microglia, possibly by suppressing the Ca2+ influx induced by ATP treatment. Finally, we recorded the spontaneous and evoked activity of spinal nociceptive-specific neuron before and after application of resting or ATP-treated microglia, with or without preincubation with a selective A1AR agonist. We found that the microglial cells, pretreated with the A1AR agonist, exhibit lower capability to facilitate the nociceptive neurons, as compared with the cells treated with ATP alone. © 2013 Wiley Periodicals, Inc.

Epigenetic therapy is a new promising area in cancer research that is based on the use of a series of molecules capable of affecting tumor cell growth, differentiation and death by modifying the cellular mechanisms underlying the control of gene expression. Significant enhancement of traditional anticancer drug effects has been also reported by several authors. Our recent research focused on the identification of new epigenetic agent-containing drug combinations to be employed in the therapy of leukemia. The results showed that the new combination of an histone deacetylase (HDAC) inhibitor and the ribonucleotide reductase (RR) inhibitor 3’-methyl-adenosine (3’-Me-Ado) is endowed with a significant antitumor activity against promyelocytic leukemia cells. This effect was dependent not only by the HDAC inhibitors-mediated enhancement of drug-induced apoptosis, but also by the ability of these pleiotropic agents to inhibit the activity of the RR enzyme. Studies are ongoing to verify the feasibility of creating new bifunctional agents containing both an RR and an HDAC inhibitor within the same molecule, so that they can reproduce the effects of this combination and furnish an easier and effective therapeutic modality in view of a possible future use in the clinics.

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.

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.