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.

Scheme 1. Chemical Structures of HL, HL^X, 1, and 1^X (X = P1–3 and M1–3)

DNA targets are highlighted by red frames.

Scheme 2. Synthetic Route of HL^X (X = P1–3 and M1–3)

Figure 1

(A) Plots of fluorescence emission intensity at 601 nm vs concentrations of 1 (red), 1^P1 (light green), 1^P2 (light blue), and 1^P3 (purple). (B) DNA binding constants K_app (×10⁶ M^–1) estimated from these data. Conditions: [complex] = 0–400 μM, [ct-DNA] = 20 μM bp, [EtBr] = 3.3 μM, [NaCl] = 10 mM, [buffer] = 10 mM (pH 6.0, MES), λ_ex = 510 nm at 37°C.

Scheme 3. Chemical Structures of 2^X (X = P1 and P3)

Figure 2

Time courses for the increase of Form III (%) in the reaction of pUC19 DNA (50 μM bp) with the complex (50 μM) and H₂O₂ (0.5 mM) at pH 6.0 (MES, 10 mM) at 37°C. (A) 1^P1 (right green), 1^P2 (right blue), 1^P3 (purple), and 1 (red) and (B) 1^M1 (right green), 1^M2 (right blue), 1^M3 (purple), and 1 (red). Experiments were repeated at least three times.

Figure 3

Plots of expected enhancement rates in DNA dsb in HeLa cells (A) × (C) shown in Table 3 vs enhancement rates in cytotoxicity against HeLa cells (B) for 1^P1–3 (red line) and 1M^1–3 (purple line). The slopes of the red and blue lines are 0.44 and 0.053, respectively.

Figure 4

Confocal microscopic images of 1^P1–3 (200 μM) in HeLa cells in the dark on 1 h incubation. Bright-field images (A, E, I). Blue fluorescence indicates the fluorescence of 1^P1 (B), 1^P2 (F), and 1^P3 (J) (λ_ex = 405 nm). Red fluorescence is mitochondrial staining by Mito Tracker Deep Red FM (50 nM) (Thermo Fisher) (C, G, K) (λ_ex = 640 nm). (D, H, L) Overlay images of panels (A)–(C), (E)–(G), and (I)–(K), respectively. The scale bar is 20 μm.