Rapid DNA interstrand cross-linking of Pt(IV) compound

Abstract

Pt(IV) anticancer compounds have been developed for several decades to overcome the drawbacks of their Pt(II) congeners, and the reduction of Pt(IV) to Pt(II) has been commonly regarded as a necessary step in the activation of Pt(IV) compounds prior to targeting DNA. However, blockage of glutathione (GSH) biosynthesis resulted in a slight effect on the cytotoxicity of oxoplatin in yeast Saccharomyces cerevisiae strains, urging us to reconsider the mechanism of actions for the "inert" Pt(IV) complexes. Using X-ray absorption near-edge spectroscopy (XANES), our data demonstrated that Pt(IV) complex oxoplatin could bind to DNA in a tetravalent state. Both alkaline denaturing agarose electrophoresis and thermal denaturation-renaturation assay revealed that oxoplatin could rapidly produce stable interstrand crosslinks (ICLs), which can further translate into a fast cell-killing process in cancer cells. Using quantitative real-time PCR and immunofluorescence analysis, we also proved that Pt(IV) complex oxoplatin could induce a quick intracellular response of the FA/BRCA pathway in cancer cells that involves the DNA interstrand crosslinking repair system, and this quick response to ICLs was independent with the intracellular GSH levels. Cell cycle analysis showed that short incubation with oxoplatin can induce a strong S phase arrest in HeLa cells, indicating that the rapid interstrand crosslinks produced by oxoplatin might stall the replication fork, result in the double-strand breaks, and eventually induce cell death. Our results implied that, besides the reduction mechanism to release the Pt(II) congeners, direct and rapid interstrand cross-linking with DNA by Pt(IV) compounds might be a unique mechanism for Pt(IV) compounds, which may provide new insight for the development of next-generation platinum-based drugs.

Keywords: DNA interstrand Cross-linking; DNA repair; FA/BRCA pathway; Pt(IV) compound; Rapid interaction.