Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) shows particular promise as an antitumour agent against colorectal cancer. It is known that KP1019 reacts with human serum proteins, whereby the major amount binds to albumin (present in large excess) and a smaller amount to transferrin. It has been hypothesised that transferrin-mediated uptake by transferrin receptor expressing tumour cells may in part explain the apparent tumour selectivity of this compound. Circular dichroism spectroscopy and electrospray ionisation mass spectrometry studies demonstrate that two equivalents of KP1019 bind specifically to human apotransferrin, while additional amounts of the ruthenium complex bind unspecifically. Uptake studies in the transferrin receptor-expressing human colon carcinoma cell line SW480 revealed a higher cellular accumulation of KP1019 in comparison to a KP1019-transferrin adduct (2:1), while the uptake of a KP1019-Fe(III)-transferrin conjugate (1:0.3:1) significantly exceeded that of KP1019, suggesting that iron binding is necessary to obtain a protein conformation which favours recognition by the transferrin receptors on the cell surface. Our study showed that KP1019 is transported into the cell by both transferrin-independent and transferrin-dependent mechanisms. Transferrin-mediated uptake is more efficient when transferrin is saturated with iron to a physiological degree (y30%). Cell fractionation experiments demonstrated that after a 2 h treatment of human colon cancer cells with 10 mM KP1019 on average 55% of the intracellular ruthenium is located in the cellular nucleus, while 45% remain in the cytosol and other cellular components.
Biotransformation of ruthenium(III) anticancer complexes as hypothesized in the activation-by-reduction theory is the central topic of the present paper. The redox behavior of tetrachlorobis(azole)ruthenate(III)-type complexes was studied by NMR spectroscopy and square wave voltammetry. The influence of reducing agents on the binding behavior toward the DNA-modeling nucleotide GMP was determined by capillary electrophoresis, accompanied by identification of arising peaks by online coupling to electrospray ionization mass spectrometry. The determination of redox potentials revealed that the biologically relevant reductants ascorbic acid and glutathione are capable of reducing the studied Ru(III) complexes under physiological conditions. Characteristic differences in reduction kinetics dependent on the pH value can be explained by higher reduction strength of ascorbic acid and glutathione at higher pH compared to the pH-independent redox response of ruthenium(III) complexes. Binding behavior of (H2ind)[trans-RuCl4(Hind)2] (Hind = 1H-indazole) toward GMP was found to be increased upon addition of two equivalents of glutathione but not of ascorbic acid. In contrast, only a minor influence on the GMP-binding under reductive conditions was found for (H2im)[trans-RuCl4(Him)2] (KP418, Him = 1H-imidazole).
Capillary electrophoresis (CE) was used as an assay for studying the interaction of (SP-4-2)-bis[(R)-(-)-2-aminobutanol)dichloroplatinum(II) (1) and (SP-4-2)bis(4-aminobutanol)dichloroplatinum(II) (2) with guanosine 5'-monophosphate (GMP). CE kinetic measurements carried out at two physiological pH levels indicated that upon increasing the pH, 1 showed an appreciable change in binding behavior, with the rate of binding increased for more than 10 times as expressed by apparent half-life values of GMP (6.1 and 62.2 h at pH 6.0 and 7.4, respectively). The rate of GMP binding for 2 remained comparatively less affected by pH (half-lives of 8.5 and 10.6 h, respectively). Regardless of the nature of platinum complex and pH, the reaction with GMP tends to be decelerated at increased chloride concentrations in solution, this effect being particularly pronounced when changing from 4 mM (intracellular level) to 100 mM (extracellular level). The kinetic differences of platinum complexes were characterized in terms of the respective GMP-adducts structure, independently identified by means of off-line electrospray ionization-mass spectrometry. Also addressed was the interpretation of binding behavior as based on the structural features of the intact complexes, namely differing inclination to intramolecular chelation.
(SP-4-2)-Bis[(R)-(-)-2-aminobutanol-kappaN]dichloroplatinum(II) and (SP-4-2)-bis[(R)-(-)-2-aminobutanolato-kappa2N,O]platinum(II) are promising cytotoxic agents exhibiting a strongly pH-dependent rate of reaction with the DNA-modeling nucleotide guanosine 5'-monophosphate (GMP). This potential mode-of-action binding, directly correlating with cytotoxicity, is influenced by the intramolecular chelation of bifunctional aminoalcohol ligands which was examined by means of micellar electrokinetic chromatography (MEKC) and nuclear magnetic resonance (NMR). While NMR clearly proves the existence of equilibrium between the ring-opened and ring-closed species, no such transformation was observed under MEKC conditions. In a kinetic study performed by MEKC, the half-lives of GMP bound to the platinum complexes were determined and compared to the kinetic data acquired by capillary zone electrophoresis. An appreciable increase in binding in the presence of sodium dodecyl sulfate (SDS) micelles was explained in terms of activation of (SP-4-2)-bis[(R)-(-)-2-aminobutanol-kappaN]dichloroplatinum(II). This apparently takes place due to the shifting of the equilibrium towards the ring-opened species, induced by adduct formation between SDS and the platinum complex that was confirmed by electrospray ionization mass spectrometry.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.