Mechanisms controlling the cellular accumulation of copper bis(thiosemicarbazonato) complexes

Copper (Cu) bis(thiosemicarbazonato) metal complexes [Cu(II)(btsc)s] have unique tumor-imaging and treatment properties and more recently have revealed potent neuroprotective actions in animal and cell models of neurodegeneration. However, despite the continued development of Cu(II)(btsc)s as potential therapeutics or diagnostic agents, little is known of the mechanisms involved in cell uptake, subcellular trafficking, and efflux of this family of compounds. Because of their high lipophilicity, it has been assumed that cellular accumulation is through passive diffusion, although this has not been analyzed in detail. The role of efflux pathways in cell homeostasis of the complexes is also largely unknown. In the present study, we investigated the cellular accumulation of the Cu(II)(btsc) complexes Cu(II)(gtsm) and Cu(II)(atsm) in human neuronal (M17) and glial (U87MG) cell lines under a range of conditions. Collectively, the data strongly suggested that Cu(II)(gtsm) and Cu(II)(atsm) may be taken into these cells by combined passive and facilitated (protein-carrier-mediated) mechanisms. This was supported by strong temperature-dependent changes to the uptake of the complexes and the influence of the cell surface protein on Cu accumulation. We found no evidence to support a role for copper-transporter 1 in accumulation of the compounds. Importantly, our findings also demonstrated that Cu from both Cu(II)(gtsm) and Cu(II)(atsm) was rapidly effluxed from the cells through active mechanisms. Whether this was in the form of released ionic Cu or as an intact metal complex is not known. However, this finding highlighted the difficulty of trying to determine the uptake mechanism of metal complexes when efflux is occurring concomitantly. These findings are the first detailed exploration of the cellular accumulation mechanisms of Cu(II)(btsc)s. The study delineates strategies to investigate the uptake and efflux mechanisms of metal complexes in cells, while highlighting specific difficulties and challenges that need to be considered before drawing definitive conclusions.