The bis-(1,10-phenanthroline)copper(II) complex Cu(phen)22+ has been widely studied regarding its biological properties, mainly due to its ability to bind to DNA and non-enzymatically cleave it. Furthermore, studies evaluating the influence of the counterion in the biological properties of different molecules have been classically reported in the literature over the years. Moreover, copper(II)-based complexes have been evaluated regarding its potential to generate reactive oxygen species (ROS) in biological environment. A recent example is the family of Casiopeinas®, more specific Casiopeina III and Casiopeina II-gly, which have advanced to clinical testing. In the search of novel agents for cancer treatment, two copper(II) compounds containing the same Cu(phen)22+ motif were synthesized varying the counterion and both showed antiproliferative activities in vitro over melanoma cells. To further evaluate its mechanism of action, agarose gel electrophoretic studies were conducted to ascertain if DNA is a possible target. This work aims to evaluate the interaction of two Cu(II) complexes containing the Cu(phen)22+ motif with DNA. Stock solutions of both complexes at 3.0 mM were initially prepared in water and further diluted. Superhelical pUC19 plasmid DNA (200 ng, 0.40 µL) was exposed to increased concentrations of both complexes (5 – 100 µM), in the presence of 2.0 µL of an 80 mM HEPES, 40 mM NaCl, pH = 7.4 buffer (1 mM of reducing agent sodium L-ascorbate was added when suitable). The total volume of each well was 20 µL. Water was used to complete the volume of the well. First well was used as control, without any complex. Mixtures were incubated at 37°C for 24 hours. To evaluate the interaction to the DNA of both Cu(phen)2Cl6TFMU and Cu(phen)2ClCl Cu(II) complexes, DNA agarose gel electrophoresis assay was conducted. At first, complexes were incubated without any reducing agent, only with DNA and buffer solutions. It was possible to identify an initial cleavage of the pUC19 supercoiled (SC) form into the open circular (OC) at 5.0 mM, been more pronounced in the following well (7.5 mM), for both complexes. At higher concentrations, the linear (L) form was also noticed (more pronounced for Cu(phen)2ClCl), which evidences a self-activating cleavage activity (metallonuclease activity in the absence of added reducing agent) for both complexes. In the next experiment, Na-L-ascorbate was added as a reducing agent, as both complexes were found to exhibit self-activating behavior. The presence of the reducing agent is intended to trigger a redox process at the copper ions, leading to the generation of reactive oxygen species (ROS) that promote further DNA damage. At a concentration of 2.0 mM, complete disappearance of DNA fragments is observed in the gel, consistent with full cleavage of pUC19 due to excessive ROS production. At lower concentrations, Cu(phen)2Cl6TFMU displays a slightly higher reducing efficiency than Cu(phen)2ClCl, as evidenced by the earlier appearance of the L form at 0.05 mM, compared to 0.50 mM for the latter. It was possible to conclude that both complexes exhibited a similar behavior regarding their interaction with pUC19 DNA, with a slightly higher reducing efficiency for Cu(phen)2Cl6TFMU. So, DNA can be considered as a possible biomolecular target for the complexes. Further studies are envisaged to confirm this hypothesis.
Almeida et al. (Sun,) studied this question.