Roles of DNA Target in Cancer Cell-Selective Cytotoxicity by Dicopper Complexes with DNA Target/Ligand Conjugates

The DNA target/ligand conjugates (HL^X, X = Pn and Mn, n = 1-3) were synthesized where various lengths of -CONH(CH₂CH₂O)CH₂CH₂NHCO- linkers with a 9-phenanthrenyl (P) or methyl (M) terminal as DNA targets replace the methyl group of 2,6-di(amide-tether cyclen)-p-cresol ligand (HL). DNA binding, DNA cleavage, cellular uptake, and cytotoxicity of [Cu₂(μ-OH)(L^X)](ClO₄)₂ (1) are examined and compared with those of [Cu₂(μ-OH)(L)](ClO₄)₂ (1) to clarify roles of DNA targets. Upon reaction of 1 with H₂O₂, μ-1,1-O₂H complexes are formed for DNA cleavage. 1, 1, and 1 are 22-, 11-, 3-fold more active for conversion of Form II to III in the cleavage of supercoiled plasmid DNA with H₂O₂ than 1, where the short P-linker may fix a dicopper moiety within a small number of base pairs to facilitate DNA double-strand breaks (dsb). This enhances the proapoptotic activity of 1, 1, and 1, which are 30-, 12-, and 9.9-fold cytotoxic against HeLa cells than 1. DNA dsb and cytotoxicity are 44% correlated in 1 but 5% in 1, suggesting specific DNA binding of P-linkers and nonspecific binding of M-linkers in biological cells. 1 exert cancer cell-selective cytotoxicity against lung and pancreas cancer and normal cells where the short P-linker enhances the selectivity, but 1 do not. Intracellular visualization, apoptosis assay, and caspase activity assay clarify mitochondrial apoptosis caused by 1. The highest cancer cell selectivity of 1 may be enabled by the short P-linker promoting dsb of mitochondrial DNA with H₂O₂ increased by mitochondrial dysfunction in cancer cells.