In vitro study of heavy metal-based anticancer complexes: activity, neurotoxicity and photon activation therapy's effect

Background. Cisplatin is one of the most efficient metal-based anticancer agents, targeting a large number of solid tumours. Despite its efficacy, cisplatin treatment is still limited by severe side effects such as neuro-, hepato- and nephro-toxicity and by resistance phenomena, only partially overcome by the use of new platinum drugs (i.e. oxaliplatin and carboplatin). These problems have stimulated the research and development of alternative therapeutic strategies based on different heavy metals. Furthermore, heavy metal-based anticancer complexes represent suitable candidates to be used in Photon Activation Therapy (PAT). Aim. In this work we investigated the in vitro activity and neurotoxicity of three anticancer complexes based on copper [Cu(PTA)4PF6], [Cu(thp)4PF6] and gold [Au(PTA)4PF6]. Furthermore we investigated the efficacy of synchrotron radiation (SR) to trigger the Auger effect in human IGROV-1 ovarian cancer cells pre-treated with [Cu(PTA)4PF6]. Material and Methods. Neurotoxicity was evaluated by an in vitro model based on the ability of NGF exposed embryonic rat dorsal root ganglia (DRG) organotypic culture to grow neurite. Furthermore the extent of proteasome inhibition in rat embryonic DRG neurons was evaluated by a fluorimetric assay. The efficacy of SR in combination with [Cu(PTA)4PF6] compared to radiation or drug alone was evaluated, in terms of cell survival, by standard clonogenic assay. DNA damage induced by the combinational treatment was also tested by RAD51 nucelar foci counting and BRCA1 nuclear expression. Results. In our model, after 48 hours of treatment, both copper-based compounds were not neurotoxic even at higher concentrations with respect to the IC50 obtained in A549 and IGROV-1 human cancer cells. On the other hand [Au(PTA)4PF6] was neurotoxic at lower concentration than IC50 calculated for cancer cell lines tested. Since the ubiquitin-proteasome system has been identified as molecular target in cancer cells for the heavy metal based-drug used in this study, we evaluated their ability to hinder the proteasome machinery in DRG neurons. Both copper-based compounds, that are not neurotoxic, do not inhibit proteasome activity in DRG neurons. On the contrary, the neurotoxic complex [Au(PTA)4PF6], induce a significant inhibition of proteasome activity even at concentration lower than the IC50. PAT experiment in IGROV-1 cells showed that the pre-treatment with [Cu(PTA)4PF6] induce an increase in cell death with respect to drug or SR alone. Furthermore the SR/[Cu(PTA)4PF6] combinational treatment induced an increase in DNA damage with respect to single treatments. Conclusions. Our results, together with the low IC50 of the copper compounds compared to the one observed for cisplatin, suggest them as promising compounds in anticancer treatment. Furthermore the neurotoxicity of the tested compounds seems to be strictly linked to a selective inhibition of proteasome activity in cancer cells with respect to DRG neurons. Finally our results suggest the potential use of [Cu(PTA)4PF6] in PAT providing support to further studies in reliable in vivo animal models.