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.