Inorganic Chemistry Communications 12 (2009) 198–200
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Uptake of VIV ion of vanadocene dichloride by apo-transferrin investigated by ESR spectroscopy Yuzo Nishida *, Aki Niinuma, Keita Abe Department of Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560, Japan
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Article history: Received 19 September 2008 Accepted 2 December 2008 Available online 24 December 2008 Keywords: Vanadocene dichloride Apo-transferrin ESR spectra Anticancer activity
a b s t r a c t The action mechanism of vanadocene dichloride, Cp2VCl2 ([email protected]
), has been investigated by interaction with apo-transferrin and albumin for its promising antitumor activities. Our ESR spectral studies clearly showed that VIV ion is readily transported from vanadocene chloride to transferrin, and the VIV ions are captured at the Fe(III) binding sites of transferrin as a VO2+ form. We also observed that some binuclear VO2+ complexes can efﬁciently transport the VO2+ ions to the apo-transferrin. Ó 2008 Published by Elsevier B.V.
Vanadocene dichloride (1) (Cp2VCl2, [email protected]
) has been shown to exhibit high antitumor activities against a wide range of murine and human tumors, with less toxic side effects than platinum antitumor agents [1–3]. Diverse biological experiments have pointed out to the nucleic acids, especially nuclear DNA, as probable primary intracellular target for vanadium-containing metabolites derived from 1, but the details of the delivery route are unclear, which should be due to the fact that the rapid hydrolysis of 1 occurs at neutral pH . Hence, the antitumor activities of 1 should be likely due to its binding to certain biomacromolecules and thus transporting to the nucleus inside tumor cells, and thus the details of the interaction between biomolecules and 1 are necessary to gain an understanding of its action mechanism. Human serum transferrin (Tf) is a single-chain glycol-protein with molecular mass of 80 kDa present in plasma at a concentration of about 35 lM, functioning a major role as iron transport . Apart from FeIII, transferrin could bind to other 30 metal ions, for example, TiIV, BiIII, and RuIII, thus leading to potential usage in diagnosis and therapy. The fact that high levels of transferrin receptors have been found on the surface of tumor cells , possibly due to their increased requirement of iron for metabolism, growth, and development, promoted us to investigate the interaction of VIV with transferrin further in order to provide a better understanding of the transport of vanadium into cells. Indeed, transferrin has already been shown to be responsible for the transport of TiIV complexes to tumor cells , and it is possible that the speciﬁc delivery of antitumor VIV complex would be achieved in a similar way. Here
* Corresponding author. Tel.: +81 23 628 4603; fax: +81 23 628 4591. E-mail address: [email protected]
(Y. Nishida). 1387-7003/$ - see front matter Ó 2008 Published by Elsevier B.V. doi:10.1016/j.inoche.2008.12.009
we will report an investigation of 1 with apo-transferrin and albumin by the use of ESR method. Proteins (apo-transferrin, albumin, and urease) were purchased from Sigma and used without further puriﬁcation, and the solutions were prepared freshly before measurements (concentration; 0.125–0.25 mM in tris–buffer solution (10 mM, pH 7.3)). Vanadocene dichloride 1 was purchased from Aldrich and all the ESR spectral measurements were performed for the solutions (tris–buffer as described above) by the use of a JEOL ESR apparatus model RE-2X (X-band at 295 K). New dark blue binuclear VO2+ complex, (VO)2(HPTP)(OH) (ClO4)2 3H2O (assumed structure being illustrated below), was obtained from the H(HPTP)4HClO4 and VOSO4, where H(HPTP) represents N,N,N0 ,N0 -tetrakis(2-pyridylmethyl)-2-hydroxy-1,3diaminopropane .
2+ N N
[(VO) 2(HPTP)(OH)]2+ Reactions of 1 and several protein solutions were studied in terms of ESR spectroscopy. Isotropic ESR spectrum of 1 in the tris–buffer solution consists of eight-line signal with appropriate
Y. Nishida et al. / Inorganic Chemistry Communications 12 (2009) 198–200
magnetic parameters (g = 1.984, |Aiso| = 6.25 mT) (Fig. 1A) . Completely identical eight-line ESR spectrum was found in the solution containing 1 and albumin (not shown, g = 1.984, |Aiso| = 6.26 mT). In the case of urease, two sets of eight-line ESR signals were observed (see Fig. 1B; species a indicated by arrow, g = 1.997, |Aiso| = 6.69 mT, species b indicated by*, g = 1.981, |Aiso| = 6.77 mT), but their magnetic parameters are apparently different from that of 1 in tris–buffer solution; this clearly demonstrates that there is no free 1 in the solution containing 1 and urease. Three sets of eight-line ESR signal were observed in the solution containing 1, albumin and urease under the same experimental conditions, implying that the third eight-line ESR signal observed in addition to the two sets of eight-line ESR signal observed in the solution containing urease and 1 cannot be attributed to free 1 in the solution. Thus, it is quite likely that almost all the VIV ion added are bound for albumin and urease, probably at the surface sites, similar to those postulated for the cases of the corresponding TiIV derivative , and the structure of VIV species at the surface sites may be similar to that proposed for in the phosphate buffer , i.e., two chloride ions of 1 are displaced by the oxygen or nitrogen atoms of the protein residues. These ESR signals disappeared after one day, which may be attributed to the formation of hydroxo- and oxo-polymers, as suggested . When 1 was added to the solution of apo-transferrin, two sets of eight-line ESR signals with different magnetic parameters have appeared as illustrated in Fig. 2A (signal a indicated by *, g = 1.985, |Aiso| = 10.3 mT; ESR parameters of the species b indicated by arrow, are essentially the same as those observed in the solution containing 1 and albumin). As the peak intensities of the species b gradually decreased as time and its ESR parameters are very similar to those observed of 1 in the albumin solution, species b may be attributed to the V(IV) species bound at the surface of the apotransferrin as described for the cases of albumin and urease. On the contrary the ESR signal of species a remains for a long time, as illustrated in Fig. 2B. As the magnetic parameters of the species a are essentially the same to those observed for the solution containing apo-transferrin and VOSO4 , but different from that of VO2+ ion in a buffer solution (g = 1.989, |Aiso| = 11.5 mT), it is quite likely that the signal a indicated by * should be due to the VIV ion
Fig. 2. ESR spectra of (A) 1 (1.33 mM) and apo-transferrin (0.17 mM), and (B) measured after 24 h.
Fig. 3. ESR spectra of (A) solution containing apo-transferrin (0.17 mM) and VOHPTP complex (0.67 mM) measured immediately after mixing, and (B) measured at 3 h after mixing.
captured at the Fe(III) binding sites of transferrin as a VO2+ form. Presence of albumin in solution does not inhibit the uptake of VIV ion by apo-transferrin. In order to gain the more information on the uptake of VIV ion by apo-transferrin, we have measured the ESR spectra of the solution containing apo-transferrin and binuclear VO2+ complex with H(HPTP) , the results being illustrated in Fig. 3. As shown, the ESR signal (g = 1.985, |Aiso| = 10.3 mT) gradually increased with time (see Fig. 3A and B). Since the magnetic parameters of the signal are essentially the same as those indicated by * in Fig. 2A, above facts are indicating that uptake of VO2+ by apo-transferrin proceeds facilely in the reaction with the VO2+-complex, and suggesting that some binuclear VO2+ complexes may exhibit high antitumor activity, because it has been pointed out that the binuclear structure of the metal chelate is essentially important to transport the metal ions to apo-transferrin [10,11].
References Fig. 1. ESR spectra of (A) 1 (2 mM solution), and (B) 1 (1.33 mM) and urease (0.2 mM).
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