The Bulk Breast Cancer Cell and Breast Cancer Stem Cell Activity of Binuclear Copper(II)-Phenanthroline Complexes

Abstract

Mononuclear copper(II)-phenanthroline complexes have been widely investigated as anticancer agents whereas multinuclear copper(II)-phenanthroline complexes are underexplored. Here the synthesis and characterisation of two new binuclear copper(II)-phenanthroline complexes 1 and 2 is reported, comprising of 2,9-dimethyl-1,10-phenanthroline or 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline, terminal chloride ligands, and bridging chloride or hydroxide ligands. The binuclear copper(II) complex containing 2,9-dimethyl-1,10-phenanthroline 1 displays nanomolar toxicity towards bulk breast cancer cells and breast cancer stem cells (CSCs) grown in monolayers, >50-fold greater than cisplatin (an anticancer metallodrug) and salinomycin (a gold-standard anti-CSC agent). Spectacularly, 1 exhibits >100-fold greater potency toward three-dimensionally cultured mammospheres than cisplatin and salinomycin. Mechanistic studies show that 1 evokes breast CSC apoptosis by elevating intracellular reactive oxygen species levels and damaging genomic DNA (possibly by an oxidative mechanism). To the best of our knowledge, this is the first study to probe the anti-breast CSC properties of binuclear copper(II)-phenanthroline complexes.

Keywords: antitumour agent; artificial metallonuclease; bioinorganic chemistry; cancer stem cell; copper.

Figure 1

Chemical structures of the binuclear copper(II)‐phenanthroline complexes 1 and 2.

Figure 2

Crystal structures of 1 (top) and 2 (bottom). Ellipsoids are set at 50 % probability level. Hydrogen atoms have been omitted for clarity, with the exceptions of atom H(1) in 2. Symmetry operation to generate equivalent atoms in 1: i=1−x, −y, 1−z.

Figure 3

(A) UV‐Vis spectrum of 1 (50 μM) in the presence of ascorbic acid (500 μM) and bathocuproine disulfonate, BCS (100 μM) in PBS : DMSO (200 : 1) over the course of 24 h at 37 °C. (B) ESI mass spectrum (positive mode) of 1 (500 μM) in H₂O : DMSO (10 : 1) in the presence of ascorbic acid (5 mM) after 24 h incubation at 37 °C.

Figure 4

(A) Quantification of mammosphere formation with HMLER‐shEcad cells untreated and treated with 1, 2, cisplatin or salinomycin at their respective IC₂₀ values for 5 days. Error bars=SD and Student t‐test, **=p<0.01. (B) Representative bright‐field images (×10) of the mammospheres in the absence and presence of 1, 2 or cisplatin at their respective IC₂₀ values for 5 days. (C) Representative dose‐response curves for the treatment of HMLER‐shEcad mammospheres with 1 or 2 after 5 days incubation. Error bars=SD.

Figure 5

(A) Copper content in whole cell, cytoplasm, nucleus, and membrane fractions isolated from HMLER‐shEcad cells treated with 1 (0.25 μM for 24 h). (B) Normalised ROS activity in untreated HMLER‐shEcad cells (control) and HMLER‐shEcad cells treated with 1 (IC₅₀ value ×2, 0.5–24 h). Error bars=SD and Student t‐test, *=p<0.05. (C) Immunoblotting analysis of γH2AX related to the DNA damage pathway. Protein expression in HMLER‐shEcad cells following treatment with 1 (0.03–0.12 μM for 24 h).

Figure 6

(A) Effect of ascorbic acid on 1‐mediated DNA cleavage after a 24 h incubation period. Lane 1: DNA only, Lane 2–3: DNA+10 and 20 μM of 1 with 10 equivalents of ascorbic acid. (B) Inhibition of 1‐mediated DNA cleavage by ^tBuOH, DMSO, KI, or NaN₃ after a 24 h incubation period. Lane 1: DNA only, Lane 2: DNA+1 (10 μM) with 10 equivalents of ascorbic acid, Lane 3–6: DNA+1 (10 μM) with 10 equivalents of ascorbic acid+^tBuOH (10 mM), DMSO (10 mM), KI (40 mM) or NaN₃ (40 mM). (C) Immunoblotting analysis of proteins related to the caspase‐dependent apoptosis pathway. Protein expression in HMLER‐shEcad cells following treatment with 1 (0.03–0.12 μM for 48 h). (D–F) FITC Annexin V‐propidium iodide binding assay plots of untreated HMLER‐shEcad cells, and HMLER‐shEcad cells treated with 1 (IC₅₀ value ×2 for 48 h) or cisplatin (25 μM for 48 h).