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

Abstract

The DNA target/ligand conjugates (HL^X, X = Pn and Mn, n = 1-3) were synthesized where various lengths of -CONH(CH₂CH₂O)_nCH₂CH₂NHCO- 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^X) are examined and compared with those of [Cu₂(μ-OH)(L)](ClO₄)₂ (1) to clarify roles of DNA targets. Upon reaction of 1^X with H₂O₂, μ-1,1-O₂H complexes are formed for DNA cleavage. 1^P1, 1^P2, and 1^P3 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^P1, 1^P2, and 1^P3, which are 30-, 12-, and 9.9-fold cytotoxic against HeLa cells than 1. DNA dsb and cytotoxicity are 44% correlated in 1^P1-3 but 5% in 1^M1-3, suggesting specific DNA binding of P-linkers and nonspecific binding of M-linkers in biological cells. 1^P1-3 exert cancer cell-selective cytotoxicity against lung and pancreas cancer and normal cells where the short P-linker enhances the selectivity, but 1^M1-3 do not. Intracellular visualization, apoptosis assay, and caspase activity assay clarify mitochondrial apoptosis caused by 1^P1-3. The highest cancer cell selectivity of 1^P1 may be enabled by the short P-linker promoting dsb of mitochondrial DNA with H₂O₂ increased by mitochondrial dysfunction in cancer cells.