Structure-activity studies, role of transferrin, and gene expression profile of the cytotoxic activity of titanium(IV) complexes in colon adenocarcinoma HT29 cells

Ligand modification on titanocene dichloride has the potential to produce soluble and stable complexes. As part of our research efforts in the area of titanium-based antitumor agents, we have investigated the cytotoxic activity of [Ti4(maltolato)8(mu-O)4], (Cp-R)2TiCl2 and (Cp-R)CpTiCl2 (R = CO 2CH3 and CO2CH2CH3), and three water soluble titanocene-amino acid complexes---[Cp2Ti(aa) 2]Cl2 (aa = L-cysteine, L-methionine, and D-penicillamine)---on the human colon adenocarcinoma cell line, HT29. Both the capacity of these complexes to donate Ti(IV) to human apo-transferrin and their hydrolytic stability have been investigated to elucidate how changes in either the ancillary ligands or the functionalized cyclopentadienyl ligands affect their water properties, the titanium intake by human apo-transferrin, and their cytotoxicity in HT29 cells. The amino acid complexes transferred Ti(IV) to human apo-transferrin at a slower rate than titanocene dichloride but eventually loaded both the N- and C-lobes. The functionalized titanocenes, (Cp-R)2TiCl 2 and (Cp-R)CpTiCl2 (R = CO2CH3 and CO2CH2CH3), undergo transference of Ti(IV) to human apo-transferrin at about the same rate as titanocene dichloride. Notably, the titanium-maltolato complex does not transferred Ti(VI) to human apo-transferrin at any time within the first seven days of its interaction, demonstrating the inert character of this species. Stability studies on these complexes showed that titanocene complexes decomposed at physiological pH while the [Ti4(maltolato)8(mu-O)4] complex was stable at this pH without any notable decomposition for a period of ten days. The antitumor activity of these complexes against colon cancer HT-29 cells was determined using an MTT cell viability assay at 72 and 96 hours. The titanocene-amino acid and the (Cp- R)2TiCl2/(Cp-R)CpTiCl 2 (R = CO2CH3) complexes were not biologically active when human transferrin was absent; they also were non-active when human transferrin was present at dose-equivalent concentrations. (Cp-R)2TiCl 2 and (Cp-R)CpTiCl2 (R = CO2 CH2CH 3) showed cytotoxic activity in HT29 cells comparable to that which is displayed by titanocene dichloride. The titanium-maltolato complex had higher levels of cytotoxic activity than any other titanocene complex investigated. Transferrin may be important in protecting the titanium center from hydrolysis, but this may be achieved by selecting ligands that could result in hydrolytically stable, yet active, complexes. The attack to nucleic acids such as DNA apparently are the main cause of the antiproliferative activity shown by titanium complexes in cancer cells, both organometallic as well as inorganic. The microarray data supported a direct DNA-damaging action for titanocene dichloride in HT29 cells, and the new titanium-maltolato complex is correlated with deregulation of genes involved in cell cycle arrest, apoptosis and autoimmune responses. This demonstrates that titanium drugs exert a true cytotoxic effect in HT29 colon adenocarcinoma cells, in which DNA damage is crucial.