Role of the NHE1 exchanger in the antitumor effects of copper(II) complexes and phenanthroline derivatives

Three copper(II) complexes containing 1,10-phenanthroline ([CuCl2(phen)]{middle dot}4H2O,1), neocuproine ([CuCl2(neo)]{middle dot}4H2O, 2) and tetramethyl-phenanthroline ([CuCl2(tmp)]{middle dot}4H2O, 3) as the primary ligand and another three copper(II) complexes with the L-Ala-Phe dipeptide as an auxiliary ligand: [Cu(L-Ala-Phe)(phen)]{middle dot}4H2O (4), [Cu(L-Ala-Phe)(neo)]{middle dot}4H2O (5) and [Cu(L-Ala-Phe)(tmp)]{middle dot}4H2O (6), inhibited cell viability in breast cancer MCF-7 cell line, both in the monolayer and spheroid cell culture models. The pair with tetramethyl-phenanthroline displayed a better selectivity index than cisPt and non-cytotoxicity-related ROS induction and apoptosis in the monolayer breast cancer model. Cell proliferation was affected by all compounds in a concentration-dependent manner, with a more substantial effect on the tetramethyl-phenanthroline complexes. Cell viability on multicellular spheroids showed a concentration-dependent reduction from 1 M, with IC50 that were half the one for cisplatin. All copper complexes, except for 1 showed DNA damage, demonstrated by the comet assay at a concentration below the IC50. The role of NHE1 has been linked to many types of cancers. Our study revealed that all compounds inhibited NHE1 activity in MCF-7 cells. However, only complexes containing the dipeptide auxiliary ligand could extend their effect on cell migration (Wound Healing Assay) and MMP-9 activity studied by zimography. Wester Blot analysis showed that expressions of MMP-2, MMP-9, and NHE1 were affected when MCF7 cells were treated with the six compounds as well. Overall, our results reveal an antitumor effect of all copper(II) complexes studied in breast cancer cells and a fundamental role of NHE1 in cell migration.