Metallohelices with activity against cisplatin-resistant cancer cells; does the mechanism involve DNA binding?
Enantiomers of a relatively rigid DNA-binding metallo-helix are shown to have comparable activity to that of cisplatin against the cell lines MCF7 (human breast adenocarcinoma) and A2780 (human ovarian carcinoma) but are ca five times more active against the cisplatin-resistant A2780cis. The cell-line HCT116 p53(+/+) (human colon carcinoma) is highly sensitive giving IC50 values in the nM range, far lower than the cisplatin control. The hypothesis that the biological target of such metallohelices is DNA is probed by various techniques. Tertiary structure changes in ct-DNA (formation of loops and intramolecular coiling) on exposure to the compounds are demonstrated by atomic force microscopy and supported by circular/ linear dichroism in solution. Selectivity for 5 '-CACATA and 5 '-CACTAT segments is shown by DNase I footprinting. Various three-and four-way oligonucleotide junctions are stabilised, and remarkably only the L metallo-helix enantiomer stabilizes T-shaped 3WJs during gel electrophoresis; this is despite the lack of a known helix binding site. In studies with oligonucleotide duplexes with bulges it is also shown for the first time that the metallo-helix binding strength and the number of binding sites are dependent on the size of the bulge. In contrast to all the above, flexible metallo-helices show little propensity for structured or selective DNA binding, and while for A2780 the cancer cell line cytotoxicity is retained the A2780cis strain shows significant resistance. For all compounds in the study, H2AX FACS assays on HCT116 p53(+/+) showed that no significant DNA damage occurs. In contrast, cell cycle analysis shows that the DNA binders arrest cells in the G2/mitosis phase, and while all compounds cause apoptosis, the DNA binders have the greater effect. Taken together these screening and mechanistic results are consistent with the more rigid helices acting via a DNA binding mechanism while the flexible assemblies do not.