Cellular and cell-free studies of catalytic DNA cleavage by ruthenium polypyridyl complexes containing redox-active intercalating ligands

Abstract

The ruthenium(ii) polypyridyl complexes (RPCs), [(phen)₂Ru(tatpp)]²⁺ (3²⁺ ) and [(phen)₂Ru(tatpp)Ru(phen)₂]⁴⁺ (4⁴⁺ ) are shown to cleave DNA in cell-free studies in the presence of a mild reducing agent, i.e. glutathione (GSH), in a manner that is enhanced upon lowering the [O₂]. Reactive oxygen species (ROS) are involved in the cleavage process as hydroxy radical scavengers attenuate the cleavage activity. Cleavage experiments in the presence of superoxide dismutase (SOD) and catalase reveal a central role for H₂O₂ as the immediate precursor for hydroxy radicals. A mechanism is proposed which explains the inverse [O₂] dependence and ROS data and involves redox cycling between three DNA-bound redox isomers of 3²⁺ or 4⁴⁺ . Cultured non-small cell lung cancer cells (H358) are sensitive to 3²⁺ and 4⁴⁺ with IC₅₀ values of 13 and 15 μM, respectively, and xenograft H358 tumors in nude mice show substantial (∼80%) regression relative to untreated tumors when the mice are treated with enantiopure versions of 3²⁺ and 4⁴⁺ (Yadav et al. Mol Cancer Res, 2013, 12, 643). Fluorescence microscopy of H358 cells treated with 15 μM 4⁴⁺ reveals enhanced intracellular ROS production in as little as 2 h post treatment. Detection of phosphorylated ATM via immunofluorescence within 2 h of treatment with 4⁴⁺ reveals initiation of the DNA damage repair machinery due to the ROS insult and DNA double strand breaks (DSBs) in the nuclei of H358 cells and is confirmed using the γH2AX assay. The cell data for 3²⁺ is less clear but DNA damage occurs. Notably, cells treated with [Ru(diphenylphen)₃]²⁺ (IC₅₀ 1.7 μM) show no extra ROS production and no DNA damage by either the pATM or γH2AX even after 22 h. The enhanced DNA cleavage under low [O₂] (4 μM) seen in cell-free cleavage assays of 3²⁺ and 4⁴⁺ is only partially reflected in the cytotoxicity of 3²⁺ and 4⁴⁺ in H358, HCC2998, HOP-62 and Hs766t under hypoxia (1.1% O₂) relative to normoxia (18% O₂). Cells treated with RPC 3²⁺ show up to a two-fold enhancement in the IC₅₀ under hypoxia whereas cells treated with RPC 4⁴⁺ gave the same IC₅₀ whether under hypoxia or normoxia.

Fig. 1. Chemical structures of RPCs and reference numbering scheme. All of these cations are soluble in water as the chloride salts.

Fig. 2. Agarose gel showing DNA cleavage products of pUC19 after treatment with RPCs 1–8 in the presence of GSH under aerobic conditions. Lane C, control showing open circular (Form II, top), linear (Form III, middle) and supercoiled (Form I, bottom) plasmid DNA. Lane 1, supercoiled plasmid DNA (144 μM DNA-bp) after 2 h incubation. Lane 2, supercoiled DNA (144 μM DNA-bp) with 120 μM GSH present after 2 h incubation. Lanes 3–7 supercoiled DNA (144 μM DNA-bp) with 12 μM RPC indicated and 120 μM GSH after 2 h incubation.

Fig. 3. Top: Relevant redox isomers of 4⁴⁺ in aqueous solution (pH 7.2). Black ball represents the [Ru(phen)₂]²⁺ fragment. Equivalent redox isomers exist for 3²⁺ with respect to the tatpp ligand. Bottom: Plot of the first reduction potential for the RPCs 1²⁺–8⁴⁺ in MeCN solvent. Open circles indicate complexes that are inactive for DNA cleavage and partially filled circles indicate complexes which cleave DNA in the presence of GSH.

Fig. 4. In vitro DNA plasmid cleavage assay in which pUC19 DNA (154 μM DNA-bp) was incubated with 4⁴⁺ (31 μM) in PBS buffer (pH 7.2) and 1.0 mM GSH at varying [O₂]. Lane 1: control, no 4⁴⁺, 220 mM O₂. Lane 2–5: DNA, 4⁴⁺, and varying amounts of O₂. Lane 4 also contains 30 μM 3,4-dihydroxybenzoate to show that this does not interfere with the assay; lane 5 contains 30 μM 3,4-dihydroxybenzoate and 5 units of protocatechuate dioxygenase.

Fig. 5. Effect of varying concentrations of SOD and catalase on the DNA cleavage activity of 3²⁺ ((A) top gel) and 4⁴⁺ ((B) bottom gel). Agarose gel (1%) stained with ethidium bromide of supercoiled pUC18 DNA (154 μM) cleavage products after incubation at 25 °C for 48 h with RPC (12.8 μM), GSH (256 μM) in 50 mM Na₃PO₄/10 mM buffer (pH 7.2). Lane 1: DNA control; lane 2: GSH and DNA; lane 3: DNA and RPC; lane 4: SOD (15 μg mL^–1) DNA; lane 5: catalase (15 μg mL^–1) and DNA; lane 6: RPC, GSH and DNA; lane 7: RPC, GSH, SOD (15 μg mL^–1) and DNA; lane 8: RPC, GSH, catalase (15 μg mL^–1) and DNA; lane 9: RPC, GSH, SOD (15 μg mL^–1), catalase (15 μg mL^–1) and DNA. All reactions were carried out under aerobic conditions.

Scheme 1

Fig. 6. H358 cells stained with DCFH-DA to image ROS production. First column is untreated cells as a negative control. Second column is the positive control where cells were dosed with 10, 20, and 30% solutions of H₂O₂ for 15 min and imaged with DCFH-DA. Third, fourth, and fifth columns show H358 cells dosed with relative IC₅₀ values of various complex as follows: 4⁴⁺ (15 μM), 3³⁺ (13 μM) and 2²⁺ (1.7 μM) for the 3 time periods indicated. DCFH-DA was then administered for 30 min and imaged using confocal microscopy (488/519 nm).

Fig. 7. Immunofluorescence staining of pATM foci in H358 cell line. The cells were fixed stained and imaged at 2, 8, and 22 h post treatment with the IC₅₀ values for 4⁴⁺ and 3²⁺.

Fig. 8. Immunofluorescence staining of γH2AX foci in H358 cell line. The cells were fixed stained and imaged at 2, 8, and 22 h post treatment with the IC₅₀ values for 4⁴⁺ and 3²⁺.

Fig. 9. Quantitative analysis of γ-H2AX foci in H358 cell line for etoposide, 4⁴⁺, and 3²⁺ using image J software. An average of 25 cells per count were used in tandem with double phase light contrast particle count.

Fig. 10. IC₅₀ of human malignant cell lines treated with RPCs 3²⁺ and 4⁴⁺ under normoxia (18% O₂) and hypoxia (1.1% O₂) represented by the blue and red bars respectively. In this case the enantiopure Δ-3²⁺ and ΔΔ-4⁴⁺ were used, which is why the IC₅₀'s reported under normoxia are lower. IC₅₀'s were determined using the MTT assay. Error bars indicate the standard deviation of IC₅₀ as measured from three 96 well plates. Each plate contained six replicates at each concentration to determine the IC₅₀.