Tài liệu Báo cáo khoa học: Covalent binding to glutathione of the DNA-alkylating antitumor agent, S23906-1 doc

Covalent binding to glutathione of the DNA-alkylating antitumor agent, S23906-1 Marie-He ´ le ` ne David-Cordonnier 1 , William Laine 1 , Alexandra Joubert 1 , Christelle Tardy 1 , Jean-Franc¸ois Goossens 2 , Mostafa Kouach 3 , Gilbert Briand 3 , Huong Doan Thi Mai 4 , Sylvie Michel 4 , Francois Tillequin 4 , Michel Koch 4 , Ste ´ phane Leonce 5 , Alain Pierre 5 and Christian Bailly 1 1 INSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, Lille, France; 2 Laboratoire de Chimie Analytique, Faculte ´ des Sciences Pharmaceutiques et Biologiques, et 3 Laboratoire de Spectrome ´ trie de Masse, Universite ´ de Lille, Lille, France; 4 Laboratoire de Pharmacognosie, Universite ´ Rene ´ Descartes (Paris 5), CNRS UMR8638, Faculte ´ des Sciences Pharmaceutiques et Biologiques, Paris, France; 5 Division Recherche Cance ´ rologie, Institut de Recherches SERVIER, Croissy sur Seine, France The benzoacronycine derivative, S23906-1, was character- ized recently as a novel potent antitumor agent through alkylation of the N2 position of guanines in DNA. We show here that its reactivity towards DNA can be modulated by glutathione (GSH). The formation of covalent adducts between GSH and S23906-1 was evidenced by EI-MS, and the use of different GSH derivatives, amino acids and dipeptides revealed that the cysteine thiol group is absolutely required for complex formation because glutathione disul- fide (GSSG) and other S-blocked derivatives failed to react covalently with S23906-1. Gel shift assays and fluorescence measurements indicated that the binding of S23906-1 to DNA and to GSH are mutually exclusive. Binding of S23906-1 to an excess of GSH prevents DNA alkylation. Additional EI-MS measurements performed with the mixed diester, S28053-1, showed that the acetate leaving group at the C1 position is the main reactive site in the drug: a reaction scheme common to GSH and guanines is presented. At the cellular level, the presence of GSH slightly reduces the cytotoxic potential of S23906-1 towards KB-3-1 epidermoid carcinoma cells. The GSH-induced threefold reduction of the cytotoxicity of S23906-1 is attributed to the reduced formation of lethal drug–DNA covalent complexes in cells. Treatment of the cells with buthionine sulfoximine, an inhibitor of GSH biosynthesis, facilitates the formation of drug–DNA adducts and promotes the cytotoxic activity. This study identifies GSH as a reactant for the antitumor drug, S23906-1, and illustrates a pathway by which GSH may modulate the cellular sensitivity to this DNA alkylating agent. The results presented here, using GSH as a biological nucleophile, fully support our initial hypothesis that DNA alkylation is the major mechanism of action of the promising anticancer drug S23906-1. Keywords: glutathione; DNA alkylation; acronycine; anti- cancer drug; mechanism of action. Introduction The alkaloid acronycine (Fig. 1) was first isolated from the bark of Acronychia baueri (also known as Sarcomeli- cope simplicifolia), a Rutaceous tree widely distributed in Australia [1,2]. This tetracyclic alkaloid was shown to be moderately cytotoxic to a wide range of tumor cells in vitro [3,4] and to display antitumor activities in vivo [5]. However, clinical testing of acronycine itself showed a poor response and the development of this compound was arrested in the early 1980s. Nevertheless, the antitumor potential of acronycine has stimulated the synthesis of numerous analogues [6–8]. Recently, the benzoacronycine derivative, S23906-1 (Fig. 1), was identified as a potent anticancer drug with activity against a variety of human tumor xenograft models in mice [9,10]. S23906-1 has been selected for advanced preclinical evaluation. From a mechanistic point of view, S23906-1 was recently characterized as a DNA alkylating agent reacting irrevers- ibly with guanine residues at the N2 position in double- stranded DNA [11]. The covalent binding to DNA is apparently responsible for the cytotoxic action [12] and the capacity of the drug to trigger apoptosis in tumor cells [13,14]. In the course of our ongoing studies aimed at characterizing the interaction of S23906-1 with biologically significant molecules, the reaction with glutathione (GSH) was examined. The observation that a tricyclic analogue of S23906-1 (i.e. 1,2-dihydroxy-1,2-dihydroacronycine diacetate) reacts covalently with benzyl mercaptan to form Correspondence to C. Bailly, INSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, 59045 Lille, France. Fax: + 33 320 16 92 29, Tel.: + 33 320 16 92 18, E-mail: bailly@lille.inserm.fr Abbreviations: BSO, buthionine sulfoximine; CD, circular dichroı ¨ sm; Cys, L -cysteine; Cys-Gly, cysteine-glycine; EI-MS, electrospray ion- ization mass spectroscopy; c-Glu-Cys, gamma-glutamic acid-cysteine; Gln, L -glutamine; c-Glu-Gly, gamma-glutamic acid-glycine; GS-DCE, S-dicarboxyethyl-glutathione; GS-Me, S-methyl-glutathi- one; GS-NO, S-nitrosoglutathione; GS-SA, glutathione sulfonic acid; GSH-O-Et, glutathione reduced ethyl ester; GSSG, oxidized gluta- thione; IC 50 , 50% inhibitory concentration; MC, mitomycin C; Met, L -methionine; N-Ac-Cys, N-acetyl- L -cysteine. (Received 10 March 2003, revised 05 May 2003, accepted 12 May 2003) Eur. J. Biochem. 270, 2848–2859 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03663.x an S-linked adduct prompted us to initiate this work [6]. GSH, which is an abundant intracellular tripeptide ( L -c- glutamyl- L -cysteinyl-glycine), was used as a model bio- nucleophile to investigate further the reactivity of S23906-1. Direct experimental evidences for the formation of covalent adducts between S23906-1 and GSH are reported here, and a reaction scheme common to DNA and GSH is described. Materials and methods Chemicals and biochemicals Synthesis of the benzoacronycine derivatives has been reported previously [7]. The enantiomers S27589-1 and S27590-1 were obtained from the racemate S23906-1 by HPLC on a cellulose stationary phase (Chiralcel OC; Chiral Technologies, Strasbourg, France). Synthesis of S28053-1 will be reported elsewhere, together with that of related mixed esters and diacid hemiesters. Reduced glutathione (GSH) and oxidized glutathione (GSSG), as well as L -cys- teine (Cys), N-acetyl- L -cysteine (N-Ac-Cys), L -methionine (Met), L -glutamine (Gln), gamma-glutamic acid-cysteine (c-Glu-Cys), cysteine-glycine (Cys-Gly), gamma-glutamic acid-glycine (c-Glu-Gly), S-methyl-glutathione (GS-Me), S-nitrosoglutathione (GS-NO), S-dicarboxyethyl-glutathi- one (GS-DCE), glutathione reduced ethyl ester (GSH-O- Et), glutathione sulfonic acid (GS-SA) and buthionine sulfoximine (BSO) were purchased from Sigma Aldrich. DNA restriction fragments The pBS plasmid was digested with PvuII and EcoRI and the resulting 117-bp fragment was labeled at the EcoRI site with [a- 32 P]dATP and avian myeloblastosis virus (AMV) reverse transcriptase. Electrophoresis on a nondenaturing 6% (w/v) polyacrylamide gel served to remove excess radioactive nucleotide, with the desired 3¢ end-labeled product being cut out of the gel and eluted overnight in 500 m M ammonium acetate/10 m M magnesium acetate and then ethanol precipitated. Gel-shift studies A typical cross-linking reaction consisted of incubating 10 lL of radiolabeled DNA, 2 lL of buffer (10 m M Na cacodylate, pH 7.0; Tris buffer was avoided owing to the presence of reactive amine functions) and 10 lL of the drug at the desired concentration in the dark at room tempera- ture, during the period specified in the legend, prior to adding 5 lL of a 50% glycerol solution containing tracking dyes. To study the inhibition of DNA alkylation, S23906-1 (50 l M ) was first incubated with an excess of GSH or derivatives (400 l M )in20lL of ammonium acetate for 1 h at 37 °C prior to the addition of the radiolabeled DNA for a further 2-h incubation period at 37 °C. DNA samples were resolved by electrophoresis under nondenaturing conditions in 6% acrylamide gels for  5 h at 300 V at room temperature in TBE buffer (89 m M Tris base/89 m M boric acid/2.5-m M Na 2 EDTA, pH 8.3). Gels were transferred to Whatman 3MM paper, dried under vacuum at 80 °C, and then analyzed on a phosphorimager (Molecular Dynamics 445SI). Circular dichro (CD) The CD spectra were obtained using a J-810 Jasco spectropolarimeter at 20 °C controlled by a PTC-424S/L Peltier type cell changer (Jasco Inc., Easton MD, USA). A quartz cell of 10-mm path length was used to obtain spectra from 450 to 290 nm with a resolution of 0.1 nm. The drug, S27590-1 (50 l M final concentration), was incubated in 1mL of 1m M ammonium acetate, pH 7.15, with or without (control) 1 m M GSH, GSSG, Cys, N-Ac-Cys, Cys-Gly, GS-Me or GS-NO (from a 100-m M stock solution previously equilibrated at pH 8.0 using KOH) in 1 m M ammonium acetate. CD spectra were collected every 10 min from 0 to 150 min. Electrospray ionization mass spectroscopy (EI-MS) The spontaneous hydrolysis of S28053-1 was monitored by EI-MS using a drug solution of 250 l M in 200 lLof1m M ammonium acetate, pH 8.0, and analyzing the sample at the various time-points specified in the figure legend. For the alkylation reactions, the various GSH derivatives (100 l M ) were incubated for 16 h at 20 °C, either alone or with 100 l M S23906-1, S27589-1, S27590-1 or S28053-1 in 200 lLof1m M ammonium acetate, pH 8.0. Samples were injected in a simple-quadruple MS API I (Perkin-Elmer Sciex) equipped with an ion-spray (nebulizer-assisted elec- trospray) source (Sciex, Toronto, Canada) using a needle prewashed with methanol. The solutions were infused continuously with a medical infusion pump (Model 11, Harvard Apparatus, South Natick, USA) at a flow rate of 5 lLÆmin )1 . Polypropylene glycol was used to calibrate the quadrupole. Ion spray mass spectra were acquired at unit resolution by scanning from m/z 200–800 with a step size of 0.1 Da and a dwell time of 2 ms. Twenty spectra were summed and recorded at an orifice voltage of +50 V, whereas the potential of the spray needle was held at +5 kV. Alkylation of plasmids and fluorescence measurements Compound S23906-1 (100 l M ) was incubated with or without increasing amounts of GSH or GSSG pre- equilibrated at pH 8.0 from 100 l M to 25 m M in 200 lL Fig. 1. Structure of the racemic cis diacetate compound, S23906-1 and the mixed ester analogue, S28053-1. Compounds S27590-1 and S27589- 1 correspond to the individual enantiomeric R,R and S,S forms of the racemate S23906-1, obtained by chiral HPLC. Ó FEBS 2003 Glutathione conjugation by S23906-1 (Eur. J. Biochem. 270) 2849 of incubation buffer (1 m M ammonium acetate) for 6 h at room temperature prior to adding 10 lg of the plasmid and a further 16 h of incubation at 37 °C. Free drug molecules were separated from DNA cross-linked molecules by phenol/chloroform/isoamyl alcohol (25 : 24 : 1) extraction followed by addition of 5 lLof5- M NaCl and precipitation of DNA using 1 mL of cold ethanol. After centrifugation at 13 800 g for 30 min, the DNA pellet was dried and dissolved in 1 mL of incubation buffer. The fluorescence of the compound covalently linked to DNA was measured using an excitation wavelength of 354 nm and an emission range from 420 to 650 nm. Spectra were recorded using a SPEX spectrofluorimeter Fluorolog. Cell culture KB-3-1 epidermoid carcinoma cells [15] were grown in DMEM (Dulbecco’s modified Eagle’s medium)-glutaMAX medium (Gibco) supplemented with 10% fetal bovine serum, penicillin (100 IUÆmL )1 ) and streptomycin (100Æ lg/mL) in a humidified atmosphere at 37 °C under 5% CO 2 . The cells were harvested by trypsinization and plated 24 h before treatment with the test drug. Survival assay The cytotoxicity of S23906-1 and the effects of GSH or derivatives on the cytotoxicity of this compound were assessed using a cell proliferation assay developed by Promega (CellTiter 96 Ò AQ ueous one solution cell prolifer- ation assay). Briefly, 3 · 10 3 exponentially growing KB-3-1 cells were seeded in 96-well microculture plates for 24 h prior to treatment with graded concentrations of S23906-1 and 1-m M GSH or derivatives, in six independent points. After 72 h of incubation at 37 °C, 20 lL of the tetrazolium dye was added to each well and the samples were incubated for a further 2 h at 37 °C. Plates were analyzed on a Labsystems Multiskan MS (type 352) reader at 492 nm. Detection of S23906-1-DNA adducts in KB-3-1 cells in the presence of GSH Approximately 1.5 · 10 6 KB-3-1 cells were grown for 24 h in 100-mm diameter dishes with 5 mL of culture medium, prior to the addition of GSH or derivatives (1 m M each) and S23906-1 (10 l M ) for 24 h. The genomic DNA was extracted from cells as described previously [11]. Briefly, after the drug treatment, cells were collected by centrifuga- tion (5 min, 188 g), washed twice with 10 mL of NaCl/P i buffer and resuspended in 2 mL of NaCl/P i containing 5m M MgCl 2 prior to the addition of 200 lLof10%SDS and mild agitation for 5 min. Proteinase K (80 lLofa 10-mgÆmL )1 stock solution) was added for a further 5 min of mild agitation, and finally 200 lLof0.1 M EDTA, pH 7.5, was added and the mixture incubated for 4 h at 37 °C. After addition of 80 lLof5 M NaCl, the DNA was extracted using 3 mL of phenol/chloroform/isoamyl alcohol (25 : 24 : 1) and centrifuged at 3000 g for 10 min, followed by two extractions with 3 mL of chloroform/isoamyl alcohol (24 : 1). Finally, the DNA was precipitated with cold ethanol and collected by centrifugation at 19 000 g for 30 min. The pellet was then dissolved in 200 lLofH 2 Oand treated for 2 h with 5 l M RNase A (10 mgÆmL )1 )toavoid RNA contamination. The DNA concentration was estimated by spectrophotometry at 260 nm. The fluores- cence of S23906-1 covalently linked to DNA was measured using a SPEX Fluorolog spectrofluorimeter with an excitation wavelength at 300 nm and an emission range from 420 to 555 nm. A similar procedure was used to detect S23906-1–DNA adducts in KB-3-1 cells previously treated using BSO. KB-3-1 cells, prepared as described above, were treated for 24 h with increasing concentrations of BSO prior to treatment with S23906-1. Genomic DNA was extracted and the fluorescence quantified as described above. Determination of the intracellular GSH content KB-3-1 cells (1 · 10 6 cells/dish) were incubated in the presence or absence of BSO for 24 h, as described above. Cells were then collected, washed with 10 mL of NaCl/P i and resuspended in 200 lLofNaCl/P i prior to lysis by freezing and thawing twice. The ThioGlo-1 TM (Calbiochem) reagent (10 l M ) was then added to the lysate (from a fresh 10-m M stock solution in dimethylsulfoxide) and the fluor- escence was immediately measured with an excitation wavelength of 360 nm and an emission wavelength of 400–650 nm. Results Molecular studies Two complementary techniques, EI-MS and CD, were deployed to study the reaction of the benzoacronycine derivative, S23906-1, with GSH. MS is particularly well suited for detecting the covalent adducts formed between S23906-1 and the tripeptide. This is shown in the mass spectrum given in Fig. 2A, with well-resolved peaks at MH + ¼ 308, 406, 448 and 490 corresponding to GSH and to the diol, mono-acetate and di-acetate forms of the parent drug, respectively. The latter three species reflect the hydrolysis of the compound in solution (see the results below with the analogue S28053-1). In addition, the incubation of S23906-1 with GSH for 16 h in 1-m M ammonium acetate yielded two species with a mass of MH + ¼ 695 and 737. They corresponded to the expected mass for the covalent GSH-drug adducts, with either one remaining acetate (MH + ¼ 737) or an OH group (MH + ¼ 695), at the C2 position. Direct evidence is given below for a reaction with GSH implicating the C1 position. These two peaks at MH + ¼ 737 and 695 provide strong evidence that the drug forms covalent complexes with the tripeptide. Similar MS experiments were performed after reacting S23906-1 with various GSH analogues differently substi- tuted or with amino acids and dipeptides each representing a portion of the L -c-glutamyl- L -cysteinyl-glycine parent compound. Typical mass spectra obtained with Cys, N-acetyl-Cys, c-Glu-Cys, Cys-Gly and GSH-O-Et are presented in Figs 2B–2F, respectively. In all cases with the compounds bearing a free SH group, we were able to detect the formation of covalent adducts with S23906-1. The reaction is particularly strong with Cys because, in this case, 2850 M H. David-Cordonnier et al. (Eur. J. Biochem. 270) Ó FEBS 2003 the Cys–drug adducts are detected readily (either the mono-acetate form at MH + ¼ 551 or the OH form at MH + ¼ 509), with very little drug remaining unbound (species at MH + ¼ 406 (diol) or 448 (monoacetate). The reaction is also very pronounced with the dipeptide Cys-Gly (Fig. 2E), whereas the level of adducts is much smaller (but still observed) with the c-Glu-Cys dipeptide (Fig. 2D). This suggests that the drug preferentially recognizes the Gly side of the tripeptide. In contrast, absolutely no covalent adducts were detected with other GSH-related compounds lacking the free thiol group. The compounds tested are listed in Table 1. The list includes the oxidized form GSSG and the S-protected GSH derivatives GS-Me, GS-NO, GS-DCE and GS-SA, but also smaller compounds such as Met, Gln and c-Glu-Gly. None of these compounds was able to react covalently with S23906-1, indicating that the SH group is strictly required for the formation of covalent adduct with the benzoacro- nycine derivative. The second method used to provide evidence for the formation of complexes between S23906-1 and GSH is CD. Because this spectroscopic approach requires optically active molecules, we did not use S23906-1 itself but the pure enantiomeric forms S27590-1 and S27589-1, which are the two cis enantiomers with the acetate groups located either above or below the plane of the aromatic chromo- phore (Fig. 1). These two compounds are equitoxic toward different tumor cell lines (data not shown) and also react equally well with DNA. This is shown in Fig. 3 from the gel- shift experiments performed with a 117-bp radiolabeled DNA substrate incubated with graded concentrations of S27589-1 or S27590-1 for 2 h (Fig. 3A) or with a single dose of each compound for up to 2 h (Fig. 3B). The band of DNA showing a retarded electrophoretic mobility reflects the formation of covalent adducts, as recently described [11]. It is clear from these kinetic experiments that the two cis enantiomers present equal capacities to react covalently with DNA. The reaction of compounds S27589-1 and S27590-1 with GSH was then monitored by CD. As shown in Fig. 4A, the CD spectrum of S27590-1 is altered upon reaction with GSH, but no change occurs with GSSG. Monitoring the changes of the CD signal at 300 nm allows us to distinguish the SH and S-protected compounds. With the former, such as GSH, Cys and Cys-Gly (open symbols in Fig. 4B), the CD amplitude at 300 nm largely decreases. With the latter, including GS-Me and GS-NO, for example (filled symbols in Fig. 4B), variations of the CD sign are extremely limited. This method thus provides an easy way to distinguish reactive (free SH) vs. nonreactive (S-protected) species. Although this technique cannot distinguish between binding and bonding, the data nicely complement the MS results to support the formation of stable complexes between GSH and the benzoacronycine derivatives. We have previously demonstrated that S23906-1 reacts covalently with DNA [11] and we now show that it also forms stable adducts with GSH. Two potential targets have thus been identified. Competition experiments were per- formed to determine whether the bonding to GSH can prevent DNA alkylation. For this purpose, the drug was first incubated for 1 h with an excess of GSH or related compounds containing a free SH group (such as GSH-O-Et, the dipeptide Cys-Gly or the amino acid Cys) or an S-protected function (such as GSSG or GS-NO) prior to addition of the 117-bp radiolabeled DNA fragment. After a 2-hreactionat37°C, the DNA samples were analyzed by electrophoresis on polyacrylamide gels. The results are displayed in Fig. 5. The alkylation of DNA is visualized by an important gel shift and the degree of retardation depends on the capacity of the drug to react with the competing GSH-related product. GSH binding to native DNA and to alkylated DNA was insignificant. Preincubation of S23906- 1 with Cys totally prevented the formation of DNA–drug adducts. An incomplete inhibition of DNA–S23906-1 complex formation was also observed with GSH and the SH-containing compounds GSH-O-Et, Cys-Gly and, to a lesser extent, c-Glu-Cys and N-Ac-Cys. It is important to note that despite the large excess of GSH compared with the drug (400-l M GSH vs. 50-l M S23906-1), the reactivity of the drug towards DNA was not completely abolished. This can be important at the cellular level. Fig. 2. EI-MS analysis of the alkylation of glutathione (GSH) and its derivatives by S23906-1. S23906-1 (100 l M ) was incubated with 100 l M GSH (M ¼ 307, panel A), L -cysteine (Cys) (M ¼ 121, panel B), N-acetyl- L -cysteine (N-Ac-Cys) (M ¼ 163, panel C), gamma-glutamic acid-cysteine (c-Glu-Cys) (M ¼ 250, panel D), cysteine-glycine (Cys- Gly) (M ¼ 178, panel E) or glutathione reduced ethyl ester (GSH- O-Et) (M ¼ 335, panel F) for 16 h at room temperature in 1 m M ammonium acetate, pH 7.15, prior to performing MS measurements in the positive ion mode. Among the different species identified for each spectrum, three species present the same molecular weight and correspond to the uncomplexed molecules: (·) the diol form (MH + ¼ 406) and (.) the mono-acetate form (MH + ¼ 448) derived from spontaneous hydrolysis of (*) the parent di-acetate form (MH + ¼ 490). Covalent binding of the drug to GSH (MH + ¼ 308) or its derivatives gives adducts where one (ß)ortwo(fl) acetate groups of S23906-1 are lost. In panel B, the peak at MH + ¼ 241 corresponds to [2xCys]H + .InpanelG,peaksatMH + ¼ 336 and 669 correspond to GSH-O-Et and [2xGSH-O-Et]H + , respectively. Ó FEBS 2003 Glutathione conjugation by S23906-1 (Eur. J. Biochem. 270) 2851 Table 1. Summary data. The Ô+Õ refers to SH-containing glutathione (GSH) derivatives which interact with S23906-1 to modify the spectrum of circular dichroı ¨ sm, form a covalent adduct identified by MS, compete with DNA in gel-shift assays, inhibit the alkylation of genomic DNA by S23906-1 and reduce the cytotoxicity of S23906-1 in the survival assay using KB-3-1 cell line. The Ô0Õ refers to the S-protected GSH derivatives which neither interact with S23906-1 nor modulate its cellular activity. +/–, Weak effect; ND, not determined. Cys, L -cysteine; Cys-Gly, cysteine-glycine; c-Glu-Cys, gamma-glutamic acid-cysteine; Gln, L -glutamine; c-Glu-Gly, gamma-glutamic acid-glycine; GS-DCE, S-dicarboxyethyl-glutathione; GS-Me, S-methyl-glutathione; GS-NO, S-nitrosoglutathione; GS-SA, glutathione sulfonic acid; GSH-O-Et, glutathione reduced ethyl ester; GSSG, oxidized glutathione; Met, L -methionine; N-Ac-Cys, N-acetyl- L -cysteine. Compound Circular dichroı ¨ sm MS Gel-shift competition Alkylation of genomic DNA Survival assay GSH + + + + + Cys + + + + + N-Ac-Cys + + + + + c-Glu-Cys ND + +/– ND + Cys-Gly + + + ND + GSH-O-Et ND + + + ND GSSG 0 0 0 0 0 Met ND 0 0 0 0 Gln ND 0 0 0 0 c-Glu-Gly ND 0 0 ND 0 GS-Me 0 0 0 0 0 GS-NO 0 0 0 0 0 GS-DCE ND 0 0 0 0 GS-SA ND 0 0 ND ND Fig. 3. Concentration- and time-dependence for the alkylation of DNA by S27589 and S27590 using electromobility shift assays. (A) The drug at the indicated micromolar concentration was incubated with a 117- bp radiolabeled DNA fragment for 2 h at room temperature prior to PAGE on a nondenaturing 6% gel. (B) The drug at a fixed concen- tration of 50 l M was reacted with DNA for up to 120 min. In both (A) and (B), experiments were performed in 1 m M sodium cacodylate, pH 7.0, and the free and bound DNA forms were separated by PAGE on nondenaturing 6% gels. Control tracks labeled ÔDNAÕ contained no drug; (b)and(f) refers to bound and free DNA, respectively. Fig. 4. Circular dichroı ¨ sm (CD) analysis of the interaction of glutathi- one (GSH) or its derivatives with the diacetate compound S27590. (A) CD spectra of S27590 (50 l M ) incubated alone (thinner line), with 1 m M GSH (broken line) or with 1 m M glutathione disulfide (GSSG) (thick line) for 90 min at 20 °C. In (B), the intensity of the CD band centered at 300 nm, characteristic of the GSH–drug complexes, is plotted vs. time. S27590 (50 l M )wasincubatedwith1-m M GSH (n), GSSG (m), L -cysteine (Cys) (s), N-acetyl- L -cysteine (N-Ac-Cys) (e), cysteine- glycine (Cys-Gly) (h), S-methyl-glutathione (GS-Me) (d)orS-meth- ylglutathione (GS-NO) (j)in1-m M ammonium acetate, pH 7.15. 2852 M H. David-Cordonnier et al. (Eur. J. Biochem. 270) Ó FEBS 2003 In sharp contrast to that observed with GSH, no inhibition of DNA alkylation was detected with the S-protected compounds, in particular GSSG, GS-NO, GS- DCE or GS-SA. This is again a strong indication that the SH group of the tripeptide is required for covalent complex formation with the benzoacronycine derivative. The fact that the inhibitory effect is significantly more pronounced with dipeptide Cys-Gly than with c-Glu-Cys suggests that the drug may establish contact with the Gly side of the peptide to react with the adjacent SH group on the central Cys residue, in agreement with the MS data presented above. These experiments indicate that the same reactive site on the drug molecule is implicated in the covalent binding to DNA and GSH. The two targets compete for the same electrophilic species. The competition between DNA and GSH was investi- gated further by fluorescence spectroscopy. The drug was incubated for 6 h with GSH or GSSG prior to addition of the DNA (in this case a supercoiled plasmid was used) and the reaction was performed for 15 h at 37 °C. Unbound drug molecules were then extracted with phenol/chloroform and the remaining DNA was precipitated with ethanol. The fluorescence spectrum of the DNA-bound S23906-1 species was then recorded. The results shown in Fig. 6A clearly reveal that the preincubation of the drug with GSSG had no effect on the capacity of the drug to alkylate DNA, whereas the incubation with GSH considerably reduced the extent of drug–DNA covalent complex formation. This is also shown in the concentration-dependence plot in Fig. 6B. A concentration of 1-m M GSH (i.e. close to the intracellular level) reduces, by about 40%, the capacity of S23906-1 to bond to DNA. At 10-m M GSH, the extent of DNA alkylation by S23906-1 is reduced by 70%. Alto- gether, the gel-shift assays (Fig. 5) and fluorescence data (Fig. 6) unambiguously reveal that the reaction of S23906-1 with GSH and DNA is mutually exclusive. Bonding to the peptide target modulates binding to the nucleic acid and vice versa. However, it should be noted that even a massive GSH concentration, such as 10 m M (exceeding the physio- logical concentration), does not completely abolish DNA alkylation. On the basis of structure–DNA alkylation and structure– activity relationships, we have recently shown that the reactive site on the S23906-1 molecule is the C1 position bearing a leaving acetate group [12]. The same reactive site must be involved in the reaction with the thiol function of GSH. To investigate this further, we performed a detailed MS analysis with an analogue of S23906-1 bearing two different ester groups at the C1 and C2 positions. The mixed diester S28053-1 (Fig. 1) bears an acetate group at C1 and a hemisuccinate group at C2, enabling a facile distinction between the two ester groups by means of MS. This compound, used here specifically to investigate the mech- anism of action of the parent compound, is as potent as S23906-1 at alkylating DNA (data not shown). Figure 7 shows the hydrolysis data for a solution of S28053-1 incubated for up to 20 h in the ammonium acetate Fig. 6. Effects of glutathione (GSH) and glutathione disulfide (GSSG) on DNA alkylation by S23906-1. (A) The fluorescence-emission spectra were recorded after incubation of S23906-1 with GSH or GSSG for 6 h at 37 °C followed by the addition of plasmid DNA (10 lg) for a further 15-h incubation period at 37 °C. The DNA was then extracted, precipitated and resuspended in buffer for measurement of fluores- cence at an excitation wavelength of 354 nm. S23906-1 (100 l M ) alone (plain line), or with 25 m M GSH (long dash) or GSSG (short dash). (B) Variation (%) of the fluorescence intensity at 520 nm for DNA-bound S23906-1 in the presence of increasing concentrations of GSH (d)or GSSG (h). The fluorescence of the drug–DNA complexes in the controls (without GSH or GSSG) was considered to be 100%. Fig. 5. Competitive binding of S23906-1 to DNA and SH-containing molecule. The drug (50 l M ) was incubated with or without glutathione (GSH) or its derivatives (400 l M each) for 1 h at 37 °C prior to the addition of the 117-bp radiolabeled DNA fragment for a further 2 h of incubation at 37 °C. The drug free (f) and bound (b)DNAformswere separated by PAGE on a 6% nondenaturing gel. Ó FEBS 2003 Glutathione conjugation by S23906-1 (Eur. J. Biochem. 270) 2853 buffer required for the MS analysis. The mass spectra recorded at 0 and 5 min correspond to a freshly made solution of S28053-1. The intact drug is, of course, predominantly found (MH + ¼ 562), but a tiny amount of the diol (MH + ¼ 406) and the mono-ester species (MH + ¼ 520 for the C2 hemisuccinate ester and MH + ¼ 448 for the C1 acetate ester) can be detected. With time, the proportions of the diol species gradually increase and, after 20 h, the diester compound S28053-1 is almost undetectable. For the two monoester species, the one bearing the acetate group is always present in a smaller amount compared to that harboring the hemisuccinate ester func- tion. It should be noted that different peaks with a mass of +14 (peaks at 406 + 14, 448 + 14 or 520 + 14, marked with different symbols in the Fig. 7) are detected and correspond to the reaction of the drug with methanol used to wash the needle of the quadrupole just prior to the injection. The asymmetric compound, S28053-1, was then incuba- ted with GSH overnight at room temperature in 1-m M ammonium acetate buffer, pH 7.15, and the resulting mixture was analyzed by MS. A typical mass spectrum is presented in Fig. 8A and the main peaks have been attributed to the different species obtained. In addition to the unreacted compounds (GSH at MH + ¼ 308.2 and S28053-1 at MH + ¼ 562) and the above mentioned hydrolysis products at MH + ¼ 406 (diol) and MH + ¼ 520 (mono hemisuccinate ester), different species corresponding to the GSH reaction products were detected. The main peak, at MH + ¼ 695.3, corresponds to the expected mass for the adduct between GSH and the drug in its alcohol form, i.e. without the ester group on the C2 position. By analogy with findings for DNA, we know that this adduct arises from the reaction of the monoester compound with GSH. A trans-esterification process con- verts the C2 ester into a C1 ester, which then immediately reacts with GSH to form the expected C2-OH adduct Fig. 7. EI-MS analysis of the hydrolysis of S28053. A250-l M drug solution prepared in 1 m M ammonium acetate, pH 7.15, was analyzed at the indicated incubation time (from 0 to 20 h in panels A–H). S28053 (MH + ¼ 562) hydrolyzes in the mono-hemisuccinate form (Ö) (MH + ¼ 520), in the mono-acetate form (s)(MH + ¼ 448), and in the diol form (h)(MH + ¼ 406). Three other peaks correspond to the reactivity of methanol (M ¼ 32) (used to wash the needle of the quadrupole) towards the mono-hemisuccinate form (MH + ¼ 534, r), the mono-acetate form (MH + ¼ 448, .)andthediolform (MH + ¼ 406, e), giving the M +14 species. Fig. 8. Alkylation reaction of S28053 with glutathione (GSH). (A) EI-MS analysis of the alkylation reaction of S28053 with GSH. A solution containing 100 l M of the drug was incubated with 100 l M GSH for 16 h at room temperature in 1 m M ammonium acetate, pH 7.15, prior to EI-MS analysis in positive ion mode. The structures of the main species identified are indicated. (B) Reaction scheme for the hydrolysis of S28053 and its reaction with GSH. The different species indicated, with the corresponding mass, are seen in the EI-MS spectra of Fig. 7 and/or Fig. 8A. 2854 M H. David-Cordonnier et al. (Eur. J. Biochem. 270) Ó FEBS 2003 (Fig. 8B). The trans-esterification pathway has been clearly demonstrated by NMR with the related mono-acetate compound [12]. This C2-OH adduct cannot derive from the diol compound, which is totally inert towards DNA [11], or with GSH (data not shown). The peak corresponding to a mass of MH + ¼ 809.5 represents the GSH-bound C2-hemisuccinate ester adduct, as depicted in the reaction scheme in Fig. 8B. Some of the smaller peaks in the mass spectrum shown in Fig. 8A remain to be attributed. Some may correspond to side reaction products after the trans- esterification process. For example, the peak at MH + ¼ 737.4 corresponds to the expected mass for the GSH-bound C2-acetate adduct. The reaction scheme presented in Fig. 8B summarizes the MS data and illustrates the hydrolysis and GSH reaction pathways for the compound S28053-1. The reactive site on the benzoacronycine derivative is the C1 position bearing the leaving acetate group. This group is absolutely essential. Its replacement with a nonleaving group (e.g. a methoxy substituent) totally prevents the reaction with the electro- philic species, be it GSH or DNA [12]. Cellular studies The reaction of S23906-1 with GSH may modulate the cellular response to the benzoacronycine derivative by quenching the alkylation of DNA, thereby decreasing the formation of potentially lethal DNA–drug covalent adducts. To test this hypothesis, we investigated the effect of GSH on drug-induced cytotoxicity using the KB-3-1 epidermoid carcinoma cell line. The cells were incubated with graded concentrations of S23906-1 for 72 h in the presence or absence of GSH, and the cytotoxic effect was measured using a conventional colorimetric assay. In these experiments, a solution containing the test drug and GSH was prepared and then added rapidly to the cellular medium. In the presence of the GSH, the cytotoxic effect was slightly reduced, by a factor of about three. The 50% inhibitory concentration (IC 50 ) value of 2.2 l M measured with S23906-1 alone was increased to 7.1 l M in the presence of 1-m M GSH. For comparison, in parallel experiments performed with the conventional N7-DNA alkylator, mechlorethamine hydrochloride, we observed a reduction of cytotoxicity by a factor of about six. In this case, the IC 50 value of 4.2 l M determined with mechlorethamine alone increased to 24.8 l M in the presence of mechlorethamine and 1 m M GSH. The tripeptide exerts a mildly negative effect on the cytotoxicity of the benzoacronycine derivative, S23906-1. If a significant proportion of the drug reacts with GSH, there should be less active drug available to alkylate the genomic DNA in cells. The amount of drug–DNA covalent complexes in cells can be estimated by fluorescence meas- urements, taking advantage of the specific fluorescence of the benzoacronycine chromophore. To evaluate the effect of GSH on the formation of DNA adducts in whole cells, KB- 3-1 cells were treated with 10 l M S23906-1 for 1 h in the presence or absence of GSH or its derivatives. The genomic DNA was subsequently isolated by phenol extraction and precipitated prior to measurement of the drug–DNA adducts by fluorescence. As shown in Fig. 9, the fluores- cence spectra of a DNA solution obtained from the KB-3-1 cells treated with S23906-1 reveal the presence of drug molecules attached to the genomic DNA. In the presence of GSH, the level of drug–DNA adducts decreased signifi- cantly, whereas it remained unchanged in the presence of GSSG. Again, an effect was observed with the SH- containing analogs, but not with the S-blocked GSH derivatives. For example, a marked decrease of the fluor- escence intensity at 500 nm was detected when cells were treated with a combination of S23906-1 and Cys. On the contrary, the association S23906-1 + c-Glu-Gly gave a level of drug–DNA adducts similar to that obtained with the drug alone. The reduced cytotoxic activity of S23906-1 in the presence of GSH can thus be explained by a decreased capacity to alkylate DNA. Indirectly, these experiments support the idea that the formation of drug–DNA covalent complexes is responsible for the cytotoxic action [12]. Finally, we evaluated the effect of BSO on the formation of drug–DNA covalent complexes in cells. BSO decreases GSH synthesis by specifically inhibiting c-glutamylcysteine synthetase [16], the rate-limiting step in GSH biosynthesis. BSO is commonly used to deplete cells in GSH, thereby potentiating the cytotoxic action of GSH-reactive anti- cancer drugs, such as melphalan and camptothecin [17]. The depleting effect of BSO in KB-3-1 cells was visualized by fluorescence using the fluorescent thiol reagent, ThioGlo- 1 TM . This maleimide derivative produces a highly fluorescent product upon its reaction with thiol groups, therefore providing a simple and sensitive assay for estimating the GSH content in cell homogenates [18]. The decrease of the fluorescence peak centered at 510 nm, observed upon adding increasing concentrations of BSO to the KB-3-1 cells, reflects the GSH-depleting effect (Fig. 10A). The cells were first incubated with BSO for 24 h prior to adding 10-l M S23906-1 and, after a further 1 h of incubation, the DNA was extracted and the level of drug–DNA adducts formed in situ was estimated by fluorescence. The fluores- cence spectra presented in Fig. 10B reveal that the number of drug–DNA complexes increases with increasing concen- trations of BSO. Depleting the cells with BSO reduces the intracellular concentration of GSH and, consequently, Fig. 9. Inhibition of the formation of S23906-1–DNA covalent com- plexes in cells in the presence of glutathione (GSH) or its derivatives. Fluorescence-emission spectra of DNA extracted from KB-3-1 epi- dermoid cells treated for 1 h with 10 l M S23906-1 or in the presence of 1-m M GSH, glutathione disulfide (GSSG), L -cysteine (Cys), N-acetyl- L -cysteine (N-Ac-Cys), glutathione reduced ethyl ester (GSH-O-Et), S-methyl-glutathione (GS-Me) or gamma-glutamic acid-glycine (c-Glu-Gly). The excitation wavelength was set at 300 nm and the emission range from 420 to 650 nm. Ó FEBS 2003 Glutathione conjugation by S23906-1 (Eur. J. Biochem. 270) 2855 permits a more pronounced alkylation of DNA. The capacity of the drug to react with the genomic DNA is inversely proportional to the concentration of GSH. The higher the GSH level, the lower the amount of drug–DNA adduct and vice versa. The increased cytotoxic effect observed in the presence of 1-m M BSO is weak (about twofold) but consistent (Fig. 10C). Altogether, these experi- ments demonstrate that GSH reduces the cytotoxic action of S23906-1 by decreasing the formation of lethal DNA– drug covalent adducts. Discussion The compound S23906-1, a diester derivative of 1,2- dihydrobenzo[b]acronycine, has been recently identified as a highly potent and promising antitumor agent [10]. The pharmacological profile of this drug is unusual in the sense that it is markedly active in orthotopic models of human solid tumors, even by the oral route, but only moderately active against murine transplantable tumors [9]. It is now well established that DNA is a potential target for this compound. We have recently demonstrated that the drug alkylates the N2 position of guanine residues exposed in the minor groove of double helical DNA [11]. Moreover, a very recent structure–activity study strongly suggests that the formation of DNA–S23906-1 covalent complexes is respon- sible for the cytotoxic action [12]. Nevertheless, the antitumor activity of a given drug rarely (and probably never) relies on the interaction with a single molecular target. The possible implication of other targets in the cytotoxic action of S23906-1 must be kept in mind. The relative intracellular abundance of GSH (0.5– 10 m M ) and the nucleophilicity of its thiolate ion prompted us to investigate its reactivity with S23906-1. The possibility that bonding of the drug to GSH decreases the extent of DNA adducts and/or other cellular nucleophiles, allowed us confirm that alkylation is the principal mechanism of action of S23906-1. The variety of experimental data reported in the present study, summarized in Table 1, fully demon- strates that the hypothesis was correct: S23906-1 does bind covalently to GSH. The complex formation requires the SH group of the Cys residue of the tripeptide. The drug reacts neither with GSSG nor with other S-blocked derivatives such as GS-NO, GS-DCE or GS-SA. A free SH group is required but it is not sufficient because not all SH- containing compounds react covalently with S23906-1. For example, additional MS analyses (not presented here) indicated that the drug does not bind covalently to dithiothreitol or the SH protein, thioredoxin. The cysteine SH group of GSH is necessary, but not sufficient, for covalent complex formation. The use of the model dipep- tides suggests that the drug preferentially recognizes the Cys-GlymoietyofGSH. The experiments performed with the human KB-3-1 carcinoma cell line indicate that GSH modulates the cellular response to S23906-1 by inhibiting DNA alkyla- tion, thereby decreasing the formation of potentially lethal DNA adducts. At first sight, this observation may have important biological implications because in vivo the drug will encounter large quantities of GSH and other thiol- containing substrates before it can reach the nucleus of tumor cells. However, despite this reactivity towards thiols, S23906-1 presents very high antitumor activities and even shows curative effects in vivo in certain tumor models [9,10]. Although covalent binding to GSH is frequently observed with DNA alkylating agents, the type of adducts formed and their biological effects often vary significantly Fig. 10. Effect of buthionine sulfoximine (BSO) on the cytotoxicity of S23906-1 and the formation of S23906-1–DNA covalent complexes in cells. (A) Effect of BSO on intracellular glutathione (GSH) contents. KB-3-1 cells were treated without (plain line) or with 0.1-, 0.25-, 0.5-, 1- or 10-m M BSO (dashed lanes) for 24 h at 37 °C prior to lysis, and the intracellular glutathione (GSH) contents were quantified using the ThioGlo-1 TM reagent. The excitation wavelength was 360 nm. The fluorescence intensity was expressed as a percentage of the control value (plain lane). (B) Fluorescence emission spectra of DNA extracted from KB-3-1 cells treated or untreated with increasing concentrations of BSO (0.1, 0.25, 0.5, 1 or 10 m M ) (dashed lines) for 24 h prior to the addition of 10 l M S23906-1 for 1 h (plain line). The excitation wave- length was set at 300 nm. The fluorescence intensity was expressed as a percentage of the control value. (C) Growth-inhibition curves for (j) S23906-1 alone and (n) S23906-1 in the presence of 1-m M BSO. KB-3- 1 cells were treated for 72 h with S23906-1 prior to measuring the viability using a conventional tetrazolium-based assay. 2856 M H. David-Cordonnier et al. (Eur. J. Biochem. 270) Ó FEBS 2003 from one drug to another. The drugs can be classified into two categories, depending on the positive or negative contribution of the GSH–drug adducts to the DNA reactivity. The first group of antitumor agents activated by reaction with GSH includes, for example, the natural product leinamycin which reacts with thiols to generate an electrophilic episulfonium ion then reacting with the N7 position of guanines in DNA [19]. This group also includes the promising anticancer drug, irofulven (6-hydroxymethyl- acylfulvene, also known as MGI 114 or HMAF), which is thought to alkylate amines in DNA. This semisynthetic derivative of illudin S is currently in phase II chemothera- peutic clinical trials for a variety of solid tumors [20,21]. Reaction of irofulven with GSH activates a diene inter- mediate for nucleophilic attack of DNA [22,23]. Another prominent member of this class of thiol-activated antitumor agents is the distamycin-a-bromoacrylic derivative PNU- 166196, designated brostallicin, which is currently under- going phase II clinical trials [24]. The cytotoxic action of this DNA minor-groove-binding agent is significantly enhanced in the presence of GSH and this effect is believed to originate from the formation of GSH–brostallicin covalent adducts [25]. S23906-1 clearly does not belongs to this group as, in the present case, the GSH–benzoacronycine adducts reduce the cytotoxic action of the drug. It should be included in the second group of compounds for which the covalent binding to GSH inactivates the antitumor agent. Similar behaviors have been reported with a variety of DNA alkylators, especially the nitrogen mustards [26,27], but these drugs have no real, inherent, affinity for DNA and thus over-expression of GSH leads to competition between concentrations of nucleophiles. In the case of S23906-1, the affinity for DNA probably pulls the competition between the DNA nucleophile and GSH towards the former. This may explain the continuing biological activity and DNA- alkylation ability of the agent, even in the presence of high levels of GSH. Covalent binding to GSH has also been observed with the drug mitomycin C (MC) [28,29]. The formation of GSH–MC conjugates competes with DNA alkylation, as is the case with S23906-1. Ternary GSH–MC– guanine N2 DNA adducts have been isolated and charac- terized [30]. The antitumor drug, cisplatinum, also reacts covalently with GSH and cysteine [31,32], as well as the pyrrolobenzodiazepine dimers, which form interstrand covalent DNA crosslinks and similarly alkylate guanines at their N2 positions in the duplex minor groove. Their cytotoxicity is also modulated by GSH and other nonpro- tein thiols, where reversible adducts are formed [33,34]. The formation of covalent adducts between S23906-1 and GSH can be viewed as a detoxification system. Considering that the intracellular concentration of S23906-1 in the tumors is probably very low, whereas the intracellular concentration of GSH is relatively high (generally >1 m M ), one can assume that the formation of GS–benzoacronycine adducts will be favored and that these adducts can be eliminated by different transporters, such as the multidrug- resistance-associated protein (MRP) which functions as a GSH S-conjugate carrier in leukemia and in lung carcinoma cells [35–37]. However, the fate of the GS-S23906-1 adducts are as yet unknown and at this stage we cannot eliminate the possibility that these glutathionyl conjugates remain capable of alkylating macromolecules and thus serve as a transport form and a reservoir for the drug. For example, the monoGSH-conjugate of cyclophophasmide can reform the tumor active metabolite 4-hydroxy-cyclophophasmide [38]. Although the GSH-S23906-1 adducts are apparently very stable and probably eliminated as such, we cannot reject the possibility that in a specific cell/tissue environment, the conjugates hydrolyze to generate a potentially alkylating drug species. More work on the fate and distribution of the GSH-conjugates of the benzoacronycine is required. More information is also needed on the reactivity of the drug towards GSH S-transferases which often participate in the development of drug resistance. The role of GSH-dependent DNA repair should also be investigated to better compre- hend the mechanism of action of S23906-1. There is also the possibility that the GSH–S23906-1 adducts detected here using an in vitro system, either do not form or form only very weakly in vivo. For example, the formation of cisplatin– glutathione adducts was found in vitro, but not in vivo after concomitant administration of cisplatin and glutathione to rats and cancer patients [39]. The primary objective of the present study was to investigate further the reactivity of S23906-1 towards the bionucleophiles DNA and GSH. The covalent binding of the drug to GSH reduced the formation of DNA adducts and slightly decreased the cytotoxic potential of the molecule. There is now little room for doubt that DNA is an important target of S23906-1 and the reaction mechan- ism clearly implicates the C1 functionality. 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It should be included in the second group of compounds for which the covalent binding to GSH inactivates the antitumor. before treatment with the test drug. Survival assay The cytotoxicity of S23906-1 and the effects of GSH or derivatives on the cytotoxicity of this compound