Ti-IV uptake and release by human serum transferrin and recognition of Ti-IV-transferrin by cancer cells: Understanding the mechanism of action of the anticancer drug titanocene dichloride

The organometallic anticancer agent titanocene dichloride, Cp2TiCl2, is now in phase II clinical trials as an anticancer drug, but its mechanism of ac tion is poorly understood. We show here that the interactions of Cp2TiCl2 w ith human serum transferrin (hTF) and that of Ti-2-hTF with adenosine triph osphate (ATP) have characteristics that could allow; transferrin to act as a mediator for titanium delivery to tumor cells. Such reactions may therefo re be important to the anticancer activity of this new class of drugs. Cp2T iCl2 reacts rapidly with human apo-transferrin under physiological conditio ns (100 mM NaCl, 25 mM bicarbonate, and 4 mM phosphate, pH 7.4) with carbon ate as a synergistic anion. The Cp ligands are released from the drug. Two- dimensional [H-1, C-13] NMR studies of epsilon-[C-13]Met-hTF show that Ti-I V loads the C-lobe first followed by the N-lobe and binds in the specific F e-III sites. The protein conformational changes induced by Ti-IV appear to be similar to those induced by Fem. Carbonate can act as a synergistic anio n in Ti-2-hTF but does not appear to be essential. A specific Ti-IV-hTF add uct is formed even in the absence of bicarbonate. When the pH of Ti-2-hTF s olutions is lowered, no Ti-IV is released at the endosomal pH of ca. 5.0-5. 5, but one Ti-IV dissociates between pH 4.5-2.0. In contrast, in the presen ce of 1 mM ATP, all Ti-IV is readily released from both lobes when the pH i s lowered from 7.0 to 4.5. Moreover, FeIII displaces Ti-IV rapidly from the C-lobe of Ti-2-hTF (<5 min) but only slowly (days) from the N-lobe. Thus, the species FeCTiN-hTF might also provide a route for Ti-IV entry into tumo r cells via the transferrin receptor. Ti-2-hTF effectively blocked cell upt ake of radiolabeled Fe-59-hTF into BeWo cells, a human placental choriocarc inoma cell line in culture. These results imply that titanium transferrin m ight be recognized by the transferrin receptor and be taken up into cancer cells.