TiIV Uptake and Release by Human Serum Transferrin and Recognition of TiIV-Transferrin by Cancer Cells: Understanding the Mechanism of Action of the Anticancer Drug Titanocene Dichloride

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Abstract
The organometallic anticancer agent titanocene dichloride, Cp2TiCl2, is now in phase II clinical trials as an anticancer drug, but its mechanism of action is poorly understood. We show here that the interactions of Cp2TiCl2 with human serum transferrin (hTF) and that of Ti2-hTF with adenosine triphosphate (ATP) have characteristics that could allow transferrin to act as a mediator for titanium delivery to tumor cells. Such reactions may therefore be important to the anticancer activity of this new class of drugs. Cp2TiCl2 reacts rapidly with human apo-transferrin under physiological conditions (100 mM NaCl, 25 mM bicarbonate, and 4 mM phosphate, pH 7.4) with carbonate as a synergistic anion. The Cp ligands are released from the drug. Two-dimensional [1H, 13C] NMR studies of ε-[13C]Met-hTF show that TiIV loads the C-lobe first followed by the N-lobe and binds in the specific FeIII sites. The protein conformational changes induced by TiIV appear to be similar to those induced by FeIII. Carbonate can act as a synergistic anion in Ti2-hTF but does not appear to be essential. A specific TiIV-hTF adduct is formed even in the absence of bicarbonate. When the pH of Ti2-hTF solutions is lowered, no TiIV is released at the endosomal pH of ca. 5.0−5.5, but one TiIV dissociates between pH 4.5−2.0. In contrast, in the presence of 1 mM ATP, all TiIV is readily released from both lobes when the pH is lowered from 7.0 to 4.5. Moreover, FeIII displaces TiIV rapidly from the C-lobe of Ti2-hTF (CTiN-hTF might also provide a route for TiIV entry into tumor cells via the transferrin receptor. Ti2-hTF effectively blocked cell uptake of radiolabeled 59Fe-hTF into BeWo cells, a human placental choriocarcinoma cell line in culture. These results imply that titanium transferrin might be recognized by the transferrin receptor and be taken up into cancer cells.