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

The ruthenium(ii) polypyridyl complexes (RPCs), [(phen)(2)Ru(tatpp)](2+) (3(2+)) and [(phen)(2)Ru(tatpp)Ru(phen)(2)](4+) (4(4+)) 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 [O(2)]. 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 H(2)O(2) as the immediate precursor for hydroxy radicals. A mechanism is proposed which explains the inverse [O(2)] dependence and ROS data and involves redox cycling between three DNA-bound redox isomers of 3(2+) or 4(4+). Cultured non-small cell lung cancer cells (H358) are sensitive to 3(2+) and 4(4+) with IC(50) 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 3(2+) and 4(4+) (Yadav et al. Mol Cancer Res, 2013, 12, 643). Fluorescence microscopy of H358 cells treated with 15 μM 4(4+) 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 4(4+) 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 3(2+) is less clear but DNA damage occurs. Notably, cells treated with [Ru(diphenylphen)(3)](2+) (IC(50) 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 [O(2)] (4 μM) seen in cell-free cleavage assays of 3(2+) and 4(4+) is only partially reflected in the cytotoxicity of 3(2+) and 4(4+) in H358, HCC2998, HOP-62 and Hs766t under hypoxia (1.1% O(2)) relative to normoxia (18% O(2)). Cells treated with RPC 3(2+) show up to a two-fold enhancement in the IC(50) under hypoxia whereas cells treated with RPC 4(4+) gave the same IC(50) whether under hypoxia or normoxia.