Kinetic Study of the Reaction of Cisplatin with Thiols

Abstract

ABSTRACT:The reactions of cisplatin [cis-diamminedichloroplatinum(II), CDDP] with glutathione (GSH) and drug thiols were investigated at 37°C in 100 mM Tris-NO 3 , pH ϳ7.4, using a clinically relevant concentration of CDDP (33 ⌴), a large excess of GSH (16.5 mM), and [NaCl] of 4.62 mM. The conditions were designed to mimic passage of CDDP through the cytosol. The reactions were studied by UV-absorption spectroscopy, high-pressure liquid chromatography (HPLC), and atomic absorption spectroscopy. CDDP 1 exerts its antitumor activity by binding to cellular DNA (Rosenberg, 1971;Gelasco and Lippard, 1999). When the drug enters the cell, it passes through the cytosol, enters the nuclear envelope, binds to nitrogen atoms on the bases of DNA, and promotes cell death by apoptosis (Bellon et al., 1991;Demarcq et al., 1994). The cytotoxic activity of CDDP thus seems to correlate with the amount of platinum (Pt) bound to DNA (Zwelling et al., 1979;Lindauer and Holler, 1996).Biological thiols such as GSH, metallothionine, and other protein thiols defend the cell against CDDP (Kraker et al., 1985;Pattanaik et al., 1992;Ishikawa and Ali-Osman, 1993;Reedijk and Teuben, 1999). Pt ions entering the cell may preferentially bind to GSH and metallothionine (Dedon and Borch, 1987), both present in millimolar concentrations in the cytoplasm (Souid et al., 1999(Souid et al., , 2001; A.-K. Souid personal observation). Because Pt-thiol adducts interact less well with DNA (Volckova et al., 2002), formation of these complexes limits the amount of drug available for binding to DNA. Furthermore, continued exposure to CDDP can increase the cytosolic sulfhydryl level (Schilder et al., 1990;Eastman 1991;Godwin et al., 1992), which may produce CDDP resistance.The sulfhydryl agents WR-1065 and mesna are often administered to mitigate toxicities of Pt-based compounds and alkylating agents, such as cyclophosphamide (Brock et al., 1982;Souid et al., 2001;Tacka et al., 2002). The protective mechanism of WR-1065 and mesna involves reaction of the thiolate ion with CDDP in a manner analogous to that of GSH (Leeuwenkamp et al., 1991;Treskes et al., 1991).Previously, we measured the rate of CDDP binding to DNA in peripheral blood mononuclear cells and ovarian cancer cells (Sadowitz et al., 2002). By blocking the cellular thiol groups and by adding known concentrations of WR-1065 or mesna, we showed how the amount of CDDP reaching the DNA is affected by thiol concentration. For example, blocking all cellular thiol groups with N-ethylmaleimide increased DNA platination by ϳ8-fold. These data were used to generate a simple kinetic model that reproduced the measured Pt bound to DNA as a function of incubation time and CDDP and thiol concentrations. The model included passage of CDDP through the cell membrane, binding of CDDP to cellular thiols, and platination of DNA. We also studied the reaction of GSH with 4-hydroperoxycyclophosphamide and acrolein ; the latter is a toxic metabolite of cyclophosphamide.Although CDDP reaction with thiols has been studied ex...

Biochemical and Proteomics Approaches to Characterize Topoisomerase IIα Cysteines and DNA as Targets Responsible for Cisplatin-Induced Inhibition of Topoisomerase IIα

“…Thus, this result is consistent with a mechanism by which cisplatin can perturb topoisomerase II function in situ. The ability of dithiothreitol to partially antagonize these effects can be explained from our spectrophotometric kinetic studies that showed that cisplatin reacted (half-time of 6 min) with dithiothreitol to form a complex similar to other sulfhydryl compounds (Ishikawa and AliOsman, 1993;Dabrowiak et al, 2002;Sadowitz et al, 2002). Using mixtures of untreated and cisplatin-treated kDNA (Fig.…”

“…These results suggest that agents that target protein sulfhydryl groups can act as either catalytic inhibitors or poisons of topoisomerase II␣. Because cisplatin is so reactive with free protein sulfhydryl groups (Ivanov et al, 1998;Dabrowiak et al, 2002;Sadowitz et al, 2002;Hagrman et al, 2003), we considered topoisomerase II␣ cysteines as possible sites responsible for the inhibition of the catalytic activity of topoisomerase II␣ observed in the presence of cisplatin. Our results using the fluorescent sulfhydryl-reactive reagent, ThioGlo-1 strongly suggested that cisplatin reacted with topoisomerase II␣ sulfhydryl groups (Fig.…”

“…Because it has been shown previously that cisplatin can react with free (Ishikawa and Ali-Osman, 1993;Dabrowiak et al, 2002) or protein sulfhydryl groups (Ivanov et al, 1998;Sadowitz et al, 2002;Hagrman et al, 2003) and because our enzyme preparation contained dithiothreitol, we first investigated the reaction of cisplatin with dithiothreitol. As shown in Fig.…”

“…However, at longer times and higher ratios, the absorbance continued to increase and after several hours a white precipitate was produced, suggesting that the initial complex formed underwent further reaction. The half-time for the reaction at 100 M of each reactant was approximately 6 min and as such is faster than its reaction with glutathione or other thiols (Ishikawa and Ali-Osman, 1993;Dabrowiak et al, 2002). Initial velocities were measured over the first 1 min of the reaction (Fig.…”

Optimization of a HPLC procedure for simultaneous determination of cisplatin and the complex cis,cis,trans-diamminedichlorodihydroxoplatinum(IV) in aqueous solutions

“…Nevertheless, for the study of cisplatin, the columns of 25 cm had been employed. [15][16][17] For larger operations, shorter columns can be used to reduce the retention time of the compounds, while Rs in not significantly affected. In this study, the use of a shorter column is not necessary because an acceptable run time of analysis is obtained (< 6 min).…”