Methyl-substituted 1,10-phenanthroline derivatives in copper(II) complexes enhance antitumor activity and inhibit NHE1 in MDA-MB-231 breast cancer cells

Three copper(II) complexes containing 1,10-phenanthroline derivatives, [CuCl₂(phen)].0.5H₂O ( 1 ), [CuCl₂(neo)].0.75H₂O ( 2 ), and [CuCl₂(tmp)].H₂O ( 3 ), were evaluated for their antitumor activity in MDA-MB-231 breast cancer cells. All complexes markedly reduced cell viability, exhibiting significantly lower IC₅₀ values and higher selectivity indices than the corresponding free ligands, CuCl₂, and cisplatin, highlighting the therapeutic advantage of metal complexation. Among them, only 1 induced DNA damage at sub-IC₅₀ concentrations.

Cytotoxicity in all complexes was associated with intracellular ROS generation and apoptosis, although 2 and 3 produced a stronger oxidative response. Complex 3 additionally promoted necrosis. Cell proliferation was inhibited in a concentration-dependent manner, accompanied by increased intracellular copper accumulation.

Moreover, 2 and 3 significantly inhibited Na⁺/H⁺ exchanger 1 (NHE1) activity, reducing cell migration and MMP-9 activity. Western blot analysis further demonstrated that all three complexes modulated the expression of NHE1, G protein-coupled estrogen receptor (GPER), and apoptosis-related proteins.

Overall, our findings show that ligand methylation enhances the antitumor activity of copper(II) complexes and shifts their mechanism of action from DNA damage-driven cytotoxicity toward ROS-mediated apoptosis, while also improving inhibition of NHE1-dependent migratory pathways. These results identify NHE1 as a key molecular target underlying the enhanced antitumor activity of methylated phenanthroline copper(II) complexes.