Báo cáo khoa học: Rhodanese–thioredoxin system and allyl sulfur compounds Implications in apoptosis induction docx

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Rhodanese–thioredoxin system and allyl sulfurcompoundsImplications in apoptosis inductionRenato Sabelli1, Egidio Iorio2, Angelo De Martino3, Franca Podo2, Alessandro Ricci2,Giuditta Viticchie`1, Giuseppe Rotilio3, Maurizio Paci1and Sonia Melino11 Department of Sciences and Chemical Technologies, University of Rome ‘‘Tor Vergata’’, Italy2 Department of Cellular Biology and Neurosciences, Istituto Superiore di Sanita`, Rome, Italy3 Department of Biological Sciences, University of Rome ‘‘Tor Vergata’’, ItalyThe induction of programmed cell death by sulfanesulfur compounds [alk(en)yl thiosulfate, selenodigluta-thione, allyl disulfide, etc.] poses significant questionsconcerning their metabolism and on the role of theenzymes involved in the cancerogenesis. Toohey [1]suggested that uncontrolled proliferation of neoplasticcells is a result of sulfane sulfur deficiency or overacti-vity of the enzymes involved in their metabolism.Several organosulfur compounds (OSCs) such asdiallyl disulfide, diallyl trisulfide, allicin and (moreKeywordsgarlic; mobile lipids; sodium 2-propenylthiosulfate (2-PTS); sulfane sulfur;sulfurtransferaseCorrespondenceS. Melino, Dipartimento di Scienze eTecnologie Chimiche, University of Rome‘‘Tor Vergata’’, via della Ricerca Scientifica00133-Rome, ItalyFax: +39 0672594328Tel: +39 0672594449E-mail: melinos@uniroma2.itE. lorio, Department of Cellular Biology andNeurosciences, Istituto Superiore di Sanita´,Viale Regina Elena, 299 00161 Rome, ItalyFax: +39 06 49387143Tel: +39 06 49902548E-mail: egidio.iorio@iss.it(Received 7 March 2008, revised 30 May2008, accepted 3 June 2008)doi:10.1111/j.1742-4658.2008.06535.xSodium 2-propenyl thiosulfate, a water-soluble organo-sulfane sulfur com-pound isolated from garlic, induces apoptosis in a number of cancer cells.The molecular mechanism of action of sodium 2-propenyl thiosulfate hasnot been completely clarified. In this work we investigated, by in vivo andin vitro experiments, the effects of this compound on the expression andactivity of rhodanese. Rhodanese is a protein belonging to a family ofenzymes widely present in all phyla and reputed to play a number of dis-tinct biological roles, such as cyanide detoxification, regeneration of iron–sulfur clusters and metabolism of sulfur sulfane compounds. The cytotoxiceffects of sodium 2-propenyl thiosulfate on HuT 78 cells were evaluated byflow cytometry and DNA fragmentation and by monitoring the progressiveformation of mobile lipids by NMR spectroscopy. Sodium 2-propenyl thio-sulfate was also found to induce inhibition of the sulfurtransferase activityin tumor cells. Interestingly, in vitro experiments using fluorescence spec-troscopy, kinetic studies and MS analysis showed that sodium 2-propenylthiosulfate was able to bind the sulfur-free form of the rhodanese, inhibit-ing its thiosulfate:cyanide-sulfurtransferase activity by thiolation of thecatalytic cysteine. The activity of the enzyme was restored by thioredoxinin a concentration-dependent and time-dependent manner. Our results sug-gest an important involvement of the essential thioredoxin–thioredoxinreductase system in cancer cell cytotoxicity by organo-sulfane sulfur com-pounds and highlight the correlation between apoptosis induced by thesecompounds and the damage to the mitochondrial enzymes involved in therepair of the Fe–S cluster and in the detoxification system.AbbreviationsDCF-DA, 2’, 7’-dichlorodihydrofluorescein diacetate dye; 2-PTS, sodium 2-propenyl thiosulfate; ESI, electrospray ionization; GSH, reducedglutathione; GSSG, oxidized glutathione; OSC, organosulfur compound; PCho, phosphocholine; RhdA(E), sulfur-free form of rhodanese;RhdA(ES), sulfur-loaded rhodanese; RhdA, recombinant rhodanese from Azotobacter vinelandii; RhdA-PS, propexylsulfide-form of rhodanese;ROS, reactive oxygen species; Trd, thioredoxin reductase; Trx, thioredoxin; TST, thiosulfate:cyanide sulfurtransferase.3884 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBSrecently), n-propyl thiosulfate and sodium 2-propenylthiosulfate (2-PTS) have been shown to suppress theproliferation of tumor cells in vitro through the induc-tion of apoptosis [2–7]. The biochemical mechanismsunderlying the antitumorigenic and antiproliferativeeffects of garlic-derived OSCs are not yet fully under-stood, although it seems likely that the rate of clear-ance of allyl sulfur groups from cells is a determinantof the overall response. It has also been hypothesizedthat a reduced or incorrect functionality of enzymesinvolved in the metabolism of OSCs may cause anexcess of seleno or sulfane sulfur compounds in thecell, inducing the onset of degenerative states andapoptosis. The in vitro study of their interaction withenzymes probably involved in the metabolism of sulfurmay explain their in vivo effects. Sulfurtransferases,enzymes that act at the borderline between sulfur andselenium metabolism, may have an important role inthe processes induced by these sulfur compounds. Inparticular, rhodanese displays a sulfurtransferase activ-ity in vitro, transferring the sulfane sulfur atom fromthiosulfate to cyanide to produce thiocyanate by adouble-displacement mechanism [thiosulfate:cyanidesulfurtransferase EC 2.8.1.1, (TST)] [8]. In this activity,rhodanese cycles between two stable intermediates,namely a sulfur-loaded form (ES) and a sulfur-freeform (E). The observed abundance of potentially func-tional rhodanese-like proteins suggests that membersof this homology superfamily (accession number:PF00581; http://sanger.ac.uk/cgi-bin/Pfam) may playdistinct biological roles [9,10]. Rhodanese modules,either alone or in combination with other proteins, canperform a variety of roles, ranging from transportmechanisms of sulfur ⁄ selenium in a biologically avail-able form [11–13], to the modulation of generaldetoxification processes [8] and to the restoration ofiron–sulfur centres in Fe–S proteins, such as ferredoxin[14]. In the last few years, many different studies havesupported the hypothesis that the rhodanese-like pro-teins have roles in ‘managing’ stress tolerance and inmaintaining redox homeostasis [15–18]. It is also note-worthy that a study, using microarray to examine thegene expression in colonic mucosa from cancerous andnormal tissues, hypothesized that a possible cause ofcolorectal cancer carcinogenesis might be located inthe mitochondria and identified the rhodanese gene asone out of three mitochondrial genes that had a statis-tically significant decrease in expression from normaltissue to tumor at every Dukes’ stage A–D [19,20].More recently an increase in TST expression wasobserved in colonocyte differentiation. In a humancolon cancer cell line, TST activity and expression weresignificantly increased by butyrate and by histone-deacetylase inhibitors, which promote the differentia-tion of these cells [21]. Thus, butyrate could protectthe colonocytes from sulfide-induced cytotoxicity, thuspromoting TST expression. [21]. Down-regulation ofrhodanese gene expression has also been observed indiseases such as Friedreich’s ataxia [22]. The associa-tion between the expression of rhodanese and degener-ative states might make rhodanese a potentialtumor ⁄ disease biomarker and treatment target. Not allcells are equally susceptible to the deleterious effects ofthe garlic sulfur compounds and, in particular, non-neoplastic cells are less susceptible [3,23]. Therefore,the greater sensitivity of tumor cells to these com-pounds may be related to a down-regulated expressionof TST, which leads to a reduced rate of clearance ofthese compounds; notably, epidemiological investiga-tion has revealed that increased consumption of garlicdiminishes the risk of stomach and colorectal cancers[24–27]. To explore the importance of rhodanese in themetabolism of the allyl-sulfur compounds, we investi-gated, in the present work, the effect of some naturalsulfur constituents of garlic on TST activity. Inparticular, we studied the interaction of 2-PTS withrhodanese and investigated the effect of this OSC onthe expression and activity of rhodanese. Sodium2-propenyl thiosulfate is present in aqueous garlicextract [28] and has an anti-aggregator effect in vitroon both canine and human platelets as a result of theinhibition of cyclooxygenase activity [29]. The anti-tumor effect of 2-PTS, resulting from induction of theapoptosis process, has been recently investigated [7].The data reported here show that 2-PTS is able tobind to the active site of rhodanese, resulting in TSTinhibition. We also investigated the role of reducedthioredoxin (Trx) as a possible sulfur acceptor in thisreaction to restore TST activity. Moreover, the cyto-toxic effect of 2-PTS on HuT 78 cells was also evalu-ated using flow cytometry analysis after treatment andby monitoring the progressive formation of mobilelipids by NMR spectroscopy. All results obtainedprovide evidence that highlights the possible roleof rhodanese in the management of the cytotoxicityof reactive OSCs in tumor cells and may contribute tothe design of a scheme of the mechanism of action of2-PTS as an apoptosis inducer.ResultsInhibition of cell cycle progression and inductionof apoptosis by 2-PTSThe effects of 2-PTS on the human T-lymphoblastoidcell line, HuT 78, were analysed and a typical time-R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compoundsFEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3885dependent and dose-dependent inhibition of cell growthof these cells as a result of the presence of 2-PTS wasobserved. In fact, a statistically significant decrease inthe number of viable cells, at 0.25, 0.5 and 1 mm concen-trations of 2-PTS and at different time-points, wasfound when comparing control and thiosulfate-treatedcells (data not shown). About 75 and 10% of the HuT78 cells were viable following exposure to 0.5 mm 2-PTSfor 24 and 48 h respectively (Fig. 1A). The growth-inhibitory effects of 2-PTS were determined by using theTrypan Blue dye-exclusion assay. Flow cytometric anal-ysis of HuT 78 cells after 24 and 48 h of treatment with0.5 mm 2-PTS, following staining with propidiumiodide, resulted in a statistically significant increase inthe fraction of subG1 compared with the control(Fig. 1B), showing a characteristic feature of apoptosis.Sodium 2-propenyl thiosulfate induced an increase inthe fraction of HuT 78 cells in the G2⁄ M phase after24 h of treatment, reaching a value of 67.43% (Fig. 1B).After 48 h, the percentage of the cell population thatshowed apoptotic features (subG1region) was $ 39%,suggesting that an antiproliferative activity of 2-PTSagainst HuT 78 cells (blockage in G2⁄ M phase) resultsin the triggering of apoptotsis.The effect of treatment with 2-PTS on DNA frag-mentation was assessed by analysis of the agarose-gelelectrophoretic pattern of HuT 78 cells (see supplemen-tary Fig. S1). A typical DNA-fragmentation pattern wasobserved as early as at 24 h after the addition of 2-PTS,thus confirming the apoptosis-inducing effects of 2-PTS.The role of reactive oxygen species (ROS) as potentialmediators of 2-PTS-induced cytotoxicity in HuT 78 cellswas also investigated. Figure 1C shows the productionof ROS during the first 3 h of treatment with 2-PTS.HuT 78 cells underwent an increase of ROS flux as earlyas 1 h after addition of 2-PTS. These results implied astrict association between 2-PTS effects and oxidativeimbalance. Reduced glutathione (GSH) is the mostimportant physiological antioxidant, both by directlyreacting with ROS and indirectly by preserving cysteineresidues of proteins from irreversible oxidations, so giv-ing rise to GS-R. The intracellular reduced (GSH) andoxidized (GSSG) glutathione levels were measured usingHPLC chromatography in order to show the potentialinvolvement of this redox system in the resistance ofHuT 78 cells to treatment with 2-PTS. Untreated cellsshowed an average intracellular GSH concentration of$ 55 nmolÆ mg)1of protein, whereas 2-PTS-treated cellsshowed a rapid and sustained increase of GSH levels(118.5 nmolÆmg)1protein) up to 12 h, probably as aresult of to the detoxification process. The intracellularGSSG content was not significantly affected by treat-ment with 2-PTS and remained at very low levels.Formation of1H-NMR-visible mobile lipids during2-PTS-induced apoptosisThe apoptotic parameters were quantitatively moni-tored by NMR spectroscopy carried out on intact HuT78 cells and on their aqueous cell extracts after cell expo-sure to 2-PTS. Detection of the progressive formation ofmobile lipids in intact cells indicated the induction ofapoptosis after treatment with 2-PTS (from 6 to 48 h).Resonances centered at d = 1.3 p.p.m., as a result ofthe saturated fatty acyl chain methylene segments-(CH2)n-, and at d = 0.9 p.p.m., arising from methylABCFig. 1. In vitro effect of 2-PTS on the growth of HuT 78 cells. (A)Viability of HuT 78 cells in culture after treatment with 0.5 mM2-PTS. Trypan Blue staining was used to differentiate viable fromnonviable cells. Data are expressed as mean ± SD. *P < 0.005,**P < 0.001 (n = 8). (B) Percentage of the cell cycle distribution ofHuT 78 cells after 24 and 48 h of treatment with 0.5 mM 2-PTS. (C)Intracellular production of ROS in HuT 78 cells after 1 and 3 h oftreatment with 0.5 mM 2-PTS, detected by measurement of DCFfluorescence using a FACSCalibur flow cytometer. Data areexpressed as mean ± SD. *P < 0.001, **P < 0.05 (n = 3).Sulfurtransferases and apoptotic natural sulfane sulfur compounds R. Sabelli et al.3886 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBSgroups, were followed. Quantitative analysis of mobilelipid spectral profiles (see the supplementary material)was obtained by measuring the peak area (a) ratioRchains= a[(CH2)n]lip⁄ a[CH3]tot, according to our previ-ous work [30], where a[(CH2)n]lipis the integral of themobile lipids, (CH2)nis the resonance and a(CH3)totisthe integral of the total CH3resonance at 0.9 p.p.m.caused by amino acids and lipids. The value Rchainsinuntreated control cells was 0.20 ± 0.1, not significantlydifferent from that measured in HuT 78 cells exposed to0.25 mm 2-PTS at any time-point. The average Rchainsvalue at 24 h of exposure to 2-PTS increased with theapoptotic fraction from 1.25 to 1.56 in cells treated with0.5 and 1.0 mm 2-PTS, respectively. The spectral profilesof cells treated with either 0.5 or 1.0 mm 2-PTS for 48 hwere mainly characterized by signals from mobile lipids(Rchains= 4.45 ± 1.1), while all resonances caused bysmall aqueous metabolites decreased to very low levelsas a result of the massive apoptosis of treated HuT 78cells, probably associated with loss of cell integrity.Quantitative analysis on1H-NMR spectra of cellextracts after exposure to 0.5 mm 2-PTS for an interme-diate time interval (24 h, not yet associated with lateapoptosis) showed that the intracellular level of taurine(measured from the triplet centred at d = 3.45 p.p.m.)decreased about two-fold from a basal control level of13.0 ± 2.4 to 6.5 ± 1.0 nmol per 106cells). Under thesame experimental conditions, individual choline-containing metabolites (tCho, detected under a reso-nance band centered at a chemical shift of$ 3.22 p.p.m.) underwent differential changes; in factphosphocholine (PCho) decreased by 45% (from15.1 ± 1.4 to 8.1 ± 0.8 nmol per 106cells), while freecholine and glycerophosphocholine both increased byabout three-fold. The changes in the levels of water-soluble choline-containing metabolites observed in cellstreated with 2-PTS for 24 h probably reflect the progres-sive activation of phospholipases and phosphodiester-ases. On the other hand, a complex network ofpathways may, in principle, contribute to the measureddecrease in PCho, a metabolite particularly sensitive toconditions determining a block in cell proliferationand ⁄ or to the activation of enzymes involved in choline–phospholipid degradation. In fact, both increases anddecreases in PCho have been reported to occur in differ-ent systems of apoptotic induction, according to partic-ular experimental conditions [30].Effect of 2-PTS on TST expression and activity inHuT 78 cellsThe effects of 2-PTS on the expression and activity ofTST in HuT 78 cells were analyzed. HuT 78 cells weretreated without and with 2-PTS at various concentra-tions (0.25, 0.5 and 1 mm). Figure 2A shows thewestern blot of the HuT 78 lysates after 8 and 24 h oftreatment with 0.5 mm 2-PTS. Densitometry measure-ments of western blots, corrected for actin or forglyceraldehyde-3-phosphate dehydrogenase (see thesupplementary material) expression, show that no sig-nificant variation of the expression of TST with respectto the control was induced by treatment with 2-PTS(see Fig. 2A). By contrast, a reduction of TST activitywas observed after 24 h, as shown in Fig. 2B.Interaction of 2-PTS and allyl compounds withrhodaneseIn vitro interaction and kinetic studies were performedusing the recombinant Azotobacter vinelandii rhoda-nese (RhdA) [31–33]. RhdA has similar properties andkinetic behaviour and a high structural homology withbovine rhodanese [34], which is the rhodanese consid-ered as an appropriate model for using to studyhuman rhodanese [32,33,35]. The A. vinelandii rhoda-ABFig. 2. Effect of 2-PTS on TST expression and activity in HuT 78cells. (A) Western immunoblotting showing the expression of TSTin HuT 78 cell lysates after treatment with 2-PTS. Thirty micro-grams of protein lysates from untreated cells (CTRL) incubated for8 and 24 h, and from cells treated with 0.5 mM 2-PTS for 8 and24 h, were analysed. A monoclonal anti-actin Ig was used as a con-trol of the protein concentrations; and densitometry measurementsof western immunoblotting were calculated by comparison withthe intensity of actin expression. (B) Percentage the TST activity ofHuT 78 cellular extracts [untreated cells (CTRL) and cells treatedwith 0.5 mM 2-PTS (2-PTS) after 8, 24 and 48 h of incubation]. Thecontrol after 24 h was used as the reference value. *P < 0.05.R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compoundsFEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3887nese has been shown to contain only one cysteine resi-due, which is essential for the catalytic reaction, andtherefore it represents a good model for using to studythe possible interaction of TST proteins with otherproteins or molecules involved in sulfur and seleniummetabolism [35,36].In order to investigate the action of 2-PTS on sulfur-transferase activity, we analyzed the effect of 2-PTS bymonitoring the changes of intrinsic fluorescence thatoccur when the rhodanese cycles between the sulfur-free [RhdA(E)] form and the sulfur-loaded [RhdA(ES)]form and that are caused by long-range energy trans-fer and local conformational changes in the protein[36–40].No fluorescence changes were observed when 2-PTSwas added to RhdA(ES) (Fig. 3A). By contrast, theaddition of 2-PTS induced an evident quenching ofintrinsic fluorescence of RhdA(E) (Fig. 3B), in a con-centration-dependent manner, indicating a specificinteraction of 2-PTS with the active site. Moreover,the quantum yield of intrinsic fluorescence of RhdA(E)after the addition of 2-PTS was lower than thatobtained following the addition of thiosulfate(DF% = 27.2 compared with thiosulfate DF% =15.7) (Fig. 3B). This major fluorescence quenching wasprobably a result of the closeness of the allyl group tothe Trp residues present in the protein active site, andis probably responsible for the differential quenchingof the intrinsic fluorescence in the two states of theenzyme [37,39,41]. Interestingly, cyanide did notrestore the unloaded form of the enzyme (Fig. 3C) anda significant loss of the TST activity of the enzymewas observed.Inactivation of sulfur-free rhodanese by 2-PTSand characterization of the derivative proteinIn order to study chemical modifications of sulfur-freerhodanese, it is necessary first to remove the persulfidefrom the enzyme by adding an excess of cyanide to theprotein, thus forming the thiocyanate product andsulfur-free rhodanese. As sulfur-free rhodanese issomewhat unstable, it is customary to add the modify-ing reagent immediately after cyanide treatment. Weperformed the experiment in this way to analyze theeffect of the 2-PTS on the sulfur-free form of theenzyme. Figure 4 shows the time course of inactivationof RhdA(E) caused by an interaction with 2-PTS. Pre-incubation of RhdA in the presence of a three-foldmolar excess of cyanide and a 200-fold molar excess of2-PTS induced a decrease of sulfurtransferase activityover time, and a complete loss of sulfurtransferaseactivity was observed after 90 min at 37 °C (Fig. 4A).The activity of treated RhdA was not restored afterdialysis, indicating that stable binding occurs between2-PTS and RhdA(E). By contrast, no inhibition of theTST activity was observed after pre-incubation ofRhdA(ES) in the presence of the same concentrationof 2-PTS without CN-. TST activity of the propenyl-sulfide-form of rhodanese, RhdA-PS, was restored bytreatment with dithiothreitol. In fact, 53.6% of theTST activity, with respect to the TST activity ofABCFig. 3. Intrinsic fluorescence changes of RhdA following the addi-tion of substrates. (A) RhdA(ES) (2.3 lM)(____), after the addition of460 lM 2-PTS (-__-) and after the addition of 460 lM CN-(- - -);(B) RhdA(E) (4.6 lM)(_____), with a molar ratio E: thiosulfate 1 : 400(- - - -), with a molar ratio E:2-PTS 1 : 200 (___) and E:2-PTS1 : 400 (__-__); and (C) RhdA(E) (5 lM)(_____), in the presence of2-PTS (E: 2-PTS 1 : 250 c ⁄ c) (___), E: 2-PTS 1 : 500 c ⁄ c(-__-) andafter the addition of CN-(E: 2-PTS: CN 1 : 500 : 1000 c ⁄ c ⁄ c) (- - - -).a.u., arbitrary units.Sulfurtransferases and apoptotic natural sulfane sulfur compounds R. Sabelli et al.3888 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBSRhdA(ES) before treatment, was recovered after30 min of incubation at room temperature (23°C) (seeFig. 4B). These results were in agreement with an oxi-dation state of the catalytic Cys. Thiosulfate sulfur-transferase activity of RhdA-PS was also restoredwhen Trx protein was added to the solution and thisactivity was dependent on the Trx concentration(Fig. 5A). Thioredoxin was able to re-establish 66% ofthe TST activity of RhdA-PS when was incubated withTrx at an RhdA-PS ⁄ Trx molar ratio of 1 : 2. The yieldof the recovery was faster and higher when thioredoxinreductase (Trd) and NADPH were also present in thesolution. Figure 5B shows the recovery of the TSTactivity of RhdA-PS in the presence of Trd, NADPH,and different molar concentrations of reduced Trx. Atotal recovery of the TST activity was obtained after70 min of incubation with 0.5 lm Trx. No increase ofthiocyanate production was observed in the presenceof Trx, Trd and NADPH without rhodanese. Theseresults, together with the fluorescence spectra, indicatean interaction of 2-PTS with the catalytic Cys. Thus,either the reduced Trx or dithiothreitol can reduce adisulfide bond between the OSC and the catalytic cys-teine of RhdA, and thereby restore the enzyme to itsactive state. This hypothesis was confirmed by MSanalysis of the new form of the enzyme. RP-HPLCchromatography of RhdA-PS and RhdA(E) was per-formed, followed by electrospray ionization (ESI) MSanalysis. Although the two forms of the proteinshowed the same retention times in RP-HPLC (see thesupplementary material), they showed different molec-ular mass peaks. In fact, RhdA-PS and RhdA(E) hadmolecular mass values that corresponded tom ⁄ z 31138.9 ± 3.19 and m ⁄ z 31063.8 ± 3, respec-tively. These results are consistent with thiolation ofABFig. 4. Inhibition of the sulfur-free form of RhdA by 2-PTS. (A)Time-dependent decrease of the TST activity of 48.3 lM RhdA(E) in50 mM Tris–HCl buffer, pH 8.0, 0.3 M NaCl. RhdA(E) was treatedwith a three-fold molar excess of cyanide and a 200-fold molarexcess of 2-PTS at 37 °C. (B) Effects of dithiothreitol on the TSTactivity of RhdA(ES) and RhdA-PS. The proteins were incubatedwith 4 mM dithiothreitol at room temperature and the TST activitywas assayed after 0, 15 and 30 min. RhdA(ES) and RhdA-PS weretreated identically, except that cyanide and 2-PTS were absent dur-ing the treatment of RhdA(ES). The TST activity of the enzymebefore treatment was taken to represent 100% activity. Each valuerepresents the average of three independent determinations. DTT,dithiothreitol.A B Fig. 5. Recovery of the TST activity of 17 lM RhdA-PS detectedusing the So¨rbo assay. (A) Recovery of TST activity after 2 h ofincubation at 25 °C in the absence and in the presence of thiore-doxin at molar ratios 1 : 0, 1 : 0.5, 1 : 1 and 1 : 2 c ⁄ c RhdA-PS ⁄ Trx.All values are expressed as a percentage of the TST activity valueof RhdA(ES). (B) Recovery of the TST activity of 8.1 lM RhdA-PSafter incubation in the presence of 0.1 U Trd, 50 lM NADPH anddifferent concentrations of Trx (0, 0.05, 0.15, 0.25 and 0.5 lM).R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compoundsFEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3889the catalytic cysteine of the enzyme with a propenylsul-fide (m ⁄ z 73), confirming modification of the protein atthe catalytic site.Sensitivity of RhdA-PS to proteolysisTo characterize the RhdA-PS form in greater detail,limited proteolysis of RhdA-PS was performed. Lim-ited proteolysis of globular proteins generally occurs atsites that contain the most flexible regions of the poly-peptide chain within a domain or at the flexible hingesbetween domains. Therefore, a limited trypsin diges-tion of RhdA-PS was performed to investigate the flex-ibility of this modified enzyme. As previouslyobserved, the sulfur-loaded form of RhdA [RhdA(ES)][42] appeared to be quite resistant to limited proteoly-sis. In fact, Rhd(ES), which was treated in the samemanner as for RhdA-PS, but in the absence of cyanideand 2-PTS, was not proteolyzed by trypsin andremained intact, even after incubation overnight(Fig. 6A). By contrast, RhdA-PS showed a higher sen-sitivity to proteolysis than RhdA(ES), as shown inFig. 6B,and a band rapid digestion was observed. Astable daughter band (b band), of about 17.3 kDa,appeared after a few minutes of proteolysis and waspresent also after many hours of digestion. These datasuggest that RhdA-PS is more flexible than RhdA(ES),probably as a result of local conformational differ-ences. RhdA-PS showed behaviour very similar to thatpreviously observed for the alkylated and oxidatedforms of the bovine rhodanese [43,44]. In fact, limitedproteolysis of the alkylated and oxidized forms yieldedfragments that were about half of the apparent molec-ular mass of the protein, as a result of cleavage at theinterdomain tether that connects the two domains intowhich the single polypeptide chain protein is folded[42,43,45]. Thus, the inactivation of the rhodanese by2-PTS induces local conformational changes that makethe enzyme much more sensitive to proteolytic degra-dation.RhdA-PS form catalysed the Trx oxidationThioredoxin oxidation analyses were performed withthe aim of clarifying the mechanism involved in restor-ing the TST activity of RhdA-PS. The oxidation ofTrx by RhdA was observed by NADPH oxidation.The change in NADPH concentration was caused byoxidation of the reduced Trx to its disulfide form,which was reduced by NADPH in the reaction cata-lyzed by Trd. The presence of RhdA(ES) at an equi-molar concentration of Trx caused oxidation of thereduced Trx at equilibrium with NADPH, as previ-ously observed for the bovine rhodanese [46,47] evenin the absence of a sulfur donor (Fig. 7A). Thus, alsoin this case, reduced Trx behaves as sulfur-acceptorsubstrate. Control experiments showed that no oxida-tion of NADPH occurred in the presence of rhodanesewhen reduced Trx was absent. The addition of 2-PTS(as a substrate of RhdA) to the solution resulted infurther oxidation of NADPH (Fig. 7A). 2-PTS wasalso able to oxidize Trx, but the presence of RhdAcaused an increase in the NADPH oxidation rate(Fig. 7A). In addition, RhdA-PS was able to catalyzeTrx oxidation. A rapid decrease in the concentrationof NADPH was observed when an equimolarABFig. 6. Time course of trypsin digestion of RhdA-PS and RhdA(ES).Three-hundred micrograms of enzyme was subjected to limiteddigestion with 1% (w ⁄ w) trypsin in 1 mL of 50 mM Tris–HCl buffer,pH 8, at room temperature. After the reaction the samples weresubjected to SDS-PAGE. (A) Lanes 2–7, proteolysis products ofRhdA(ES) (lane 1) at 0, 5, 10, 15, 20 and 30 min and overnight incu-bation. (B) Lanes 2–8, proteolysis products of RhdA-PS (lane 1) at0, 5, 10, 15, 20, 30, 60 min and overnight incubation, respectively.‘a’ and ‘b’ bands are the parent and daughter bands, at about 29.7and 17.3 kDa respectively. Molecular markers are on the left. STDs,molecular mass standards.Sulfurtransferases and apoptotic natural sulfane sulfur compounds R. Sabelli et al.3890 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBSconcentration of RhdA-PS was added to the stabilizedsolution containing Trx, 0.1 U Trd and 50 lm NADPH(Fig. 7B). Moreover, the subsequent addition of 2-PTSto the solution led to a further increase in NADPHoxidation. These results indicate that the rhodanese–Trx–Trd system is not inhibited by 2-PTS and that thissystem has an antioxidant action against OSCs as wella sulfane sulfur detoxification system.DiscussionNatural compounds, which improve detoxificationenzymes and ⁄ or reduce the expression and activity ofthe carcinogen activating enzymes, are good candidatesfor cancer chemoprevention. The controlled prolifera-tion of the cell may be a result of the presence of a sul-fane sulfur compound that has the ability to reduce orpromote the activity of important proteins implicatedin the cellular process. Many OSCs present in alliumvegetables have been shown to be able to inhibit theproliferation of cancer cells [4,48–51]. The induction ofprogrammed cell death by sulfane sulfur compoundsraises relevant questions about the role of enzymesinvolved in their metabolism. A feature of a neoplasticcell is the residual activity of cysteine aminotransfer-ase, 3-mercaptopyruvate sulfurtransferase and rhoda-nese, as well as the total lack of cystathionase activity[52]; in fact, the biosynthesis and transport of com-pounds from the sulfane sulfur pool does not occur inthese cells [53].Our studies presented here, on HuT 78 cells, showedthat the viability of these cells is reduced significantlyupon 24 h of exposure to 2-PTS and that this reducedgrowth rate was related to a blockage in the G2⁄ Mphase of the cell cycle.The detection, by NMR, of increasing amounts ofmobile lipids in 2-PTS-treated HuT 78 cells, furthersupports mitochondrial dysfunction in these cells. Infact, several studies have reported the appearance ofmobile lipids in a variety of cells induced to apoptosis[33,54–56], in which the characteristic apoptotic pheno-type generally results in loss of the mitochondrialmembrane potential, release of cytochrome c and mito-chondrial-dependent activation of effector caspases.Recently, alterations of mitochondrial functions bydifferent uncouplers of the respiratory chain have beenfound to be responsible for the accumulation of intra-cellular lipid bodies and therefore for the detection ofNMR-visible mobile lipids in intact HuT 78 cells (E.lorio, C. Testa, A. Stringaro, M. Condello, G. Ara-ncia, E. Lococo, R. Carnevale, R. Strom, L. Lenti &F. Podo, unpublished data). Furthermore, mobile lipidsignals have been reported in tumor cells exposed tolipophilic cationic compounds that cause mitochon-drial damage [57]. Also the observed decrease of intra-cellular taurine in cells exposed to 2-PTS for 24 h (i.e.before the occurrence of massive cell death at 48 h)seems to be in general agreement with the view of pro-gressive mitochondrial impairment in these cells. Infact, Hansen et al. [58] recently suggested a new role oftaurine in mitochondrial function by acting as a modu-lator of pH in the mitochondrial matrix and thereforealtering the overall oxidative capacity of this subcellu-lar organelle, including a reduction in fatty acyl b-oxi-dation. According to this hypothesis, the simultaneousdecrease in taurine and an increase in NMR-detectedmobile lipids suggest a substantial 2-PTS-induced lossof mitochondrial function with subsequent accumula-tion of long fatty acyl chains in triglycerides in cyto-plasmatic lipid bodies. In recent years, it has becomeA B Fig. 7. Trx oxidation by 2-PTS, in the presence and in the absenceof rhodanese, by measurement of NADPH (50 lM) consumption(absorbance at 340 nm), in 50 mM Tris–HCl buffer, pH 8.0, 1 mMEDTA, with 4 lM Trx, 0.1 U Trd and in the presence of 0.5 mM2-PTS. (A) Tris–HCl buffer (a) or 4 lM RhdA(ES) (b) was added tothe solution after 30 min at 37 °C, and, after stabilization, 2-PTSwas added. The data were normalized against an A340of 0.093(which represents 100%). (B) Tris–HCl buffer (a) or 4 lM RhdA-PS(b) was added to the solution after 15 min of incubation at 37 °Cand, after stabilization (about 10 min), 2-PTS was added. The datawere normalized against an A340of 0.085 (which represents100%).R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compoundsFEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3891apparent that mitochondria are integrally involved inthe mechanism of cell death, and anticancer drugs,which inhibit the functions of mitochondria can sensi-tize the cells to undergo apoptosis [59,60]. It is impor-tant to consider that rhodanese is an importantmitochondrial enzyme in mammalian cells [61] and,certainly, the rhodanese–Trx system is involved in theintegrity of this main energy-generator organelle.It is noteworthy that recently it has also been dem-onstrated that rhodanese deficiency affects the activityof Fe–S enzymes of the tricarboxylic acid cycle, suchas the aconitase in A. vinelandii [15]. These studies sug-gest that in tumor cells in which there is a decrease inexpression of the TST gene [19,20], the involvement ofrhodanese in the detoxification of OSCs could lead toan inhibition of the normal function of the cyanidedetoxification and Fe–S repair activities of thisenzyme, thus inhibiting the normal functions of themitochondria. The elucidation of interactions betweenOSCs and proteins, such as rhodanese or Trx that areinvolved in maintaining the redox homeostasis of thecell, could help to explain the mechanism whereby theycan induce programmed cell death in tumor cells. Inthe present work we investigated the effect of 2-PTSon the activity and expression of TST. We performedour experiments in vitro using the A. vinelandii rhoda-nese. The data reported here demonstrate that 2-PTSis able to interact with the Cys of RhdA(E), inducinga covalent modified form (RhdA-PS). 2-PTS interactswith the active site of the rhodanese enzyme by thiola-tion of the catalytic cysteine, therefore inhibiting itsTST activity. A peculiar characteristic of the A. vine-landii enzyme is the presence of only one cysteine resi-due, which is the catalytic site. The structuralsimilarity of RhdA with the bovine rhodanese led usto speculate that 2-PTS can induce a thiolation at thelevel of the catalytic Cys residue of the human rhoda-nese. The ability of this garlic compound to thiolate aninternal Cys, such as that of the active site of rhoda-nese, is an important observation that should be bornein mind when considering the mechanism of action ofOSCs. We showed that the thiolation of a mitochon-drial enzyme, which is involved in ‘managing’ theredox state of the cell, could be a relevant event in themechanism of action of this compound.The major sensitivity of RhdA-PS to degradation byproteolysis is an important factor that should be con-sidered. Limited proteolysis of RhdA-PS shows that,as well the alkylation of bovine rhodanese [43], localconformational changes occur when the enzyme ismodified by 2-PTS interactions, and high flexibility ofthe enzyme with respect to RhdA(ES) is induced.These data were also in agreement with a significantdecrease in the expression of TST after 48 h of treat-ment with 2-PTS (data not shown). Therefore, themajor flexibility of RhdA-PS may also explain itsability to interact with and oxidize the Trx comparedto RhdA(ES).Our results highlight a direct interaction betweenOSCs from garlic and rhodanese. The bond of thepropenyl sulfide with the catalytic cysteine shows thecharacteristic of a disulfide bond (i.e. it is not cleavableby nucleophilic attack of the cyanide). The propenyl-sulfur protein has a low redox potential, so Trx anddithiothreitol reduce it to restore the TST activity.These results provide evidence that, in vitro, reducedTrx also regulates TST activity via redox regulation.2-PTS alone was also able to oxidize the reduced Trx,but in the presence of RhdA(ES) or RhdA-PS Trx wasoxidized more rapidly. The results presented here sup-port a possible mechanism (Fig. 8) where the reducedTrx could be the sulfide acceptor of the rhodanese in theintracellular system. Trd was able to reduce the oxidizedTrx, and thus this process may be a potential sulfanesulfur detoxification system present in the cell. Thisresult highlights the antioxidant action of a rhodanese–Trx–Trd system against OSCs. As described in previouswork, reduced Trx is a sulfur-acceptor substrate for rho-danese. It has been also hypothesized that a primaryfunction of the rhodanese could be Trx-linked oxygenradical detoxification [46,47]. The data presented hereshed new light on the role of this enzyme in the sulfanesulfur detoxification system and on the involvement ofthe Trx–Trd system in this process. Recently, evidencefor the down-regulation of TST expression in somecancer cells has been reported [20,21]. Our data showthat no change in the expression of rhodanese occursafter 8 and 24h of treatment with 2-PTS. By contrast,O||HC2HC-HC=2O-S-S--aN+dhR-S__||OSTP-2S__S__|HC2dhRHS53xrTS23-S23__S__HC2__HC=HC2xrTS53__HdhR-S__2|HC||HC2HPDANdTxrTS53S23|SH__HC2__HC=HC2TrdFig. 8. Scheme of the proposed reactions of the interactionbetween rhodanese (Rhd) and 2-PTS, and of the restoration of rho-danese activity by the thioredoxin (Trx)–thioredoxin reductase (Trd)system in the cell.Sulfurtransferases and apoptotic natural sulfane sulfur compounds R. Sabelli et al.3892 FEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBSa significant reduction of TST activity was observed dur-ing treatment, indicating that cyanide detoxification ofrhodanese was reduced by the presence of 2-PTS. Thus,the data suggest that rhodanese could be a targetenzyme of the garlic OSCs and that the reduced TSTactivity could be caused by an increase of the sulfur-detoxification activity of the enzyme, which alsoinvolves the Trx system.The ability of 2-PTS to inhibit, either in vitro or inthe cell, the TST activity of the rhodanese and to oxi-dize the Trx in vitro, both in the absence and in thepresence of rhodanese, can be related to its ability toinduce apoptosis in the cell. Our studies showed thatthe viability of HuT 78 cells is reduced significantlyafter 24 h of exposure to 2-PTS and this reducedgrowth rate is related to a blockage in the G2⁄ M phaseof the cell cycle. HuT 78 cells underwent an earlyincrease of ROS flux after the addition of 2-PTS, and2-PTS-treated cells showed a rapid and sustainedincrease of GSH levels up to 12 h, most probablybecause of a detoxification process. These resultsimplied a strict correlation between the apoptoticeffects of 2-PTS and oxidative imbalance, and this canalso be linked to a reduction in the ability of the rhoda-nese–Trx system to detoxify by oxygen radicals in vivo.Interestingly, the blockage in the G2⁄ M phase of thecell cycle was linked to an early increase of ROS flux.Cells treated with 2-PTS showed a rapid and sustainedincrease in GSH levels up to 12 h; this was attributableto a detoxification process. These results imply a strictcorrelation between 2-PTS apoptotic effects and oxida-tive unbalance, and this can be also linked to a reduc-tion in the activity of the oxygen radicals detoxificationof the rhodanese–thioredoxin system.These results are in agreement with previous studiesreported by Chang et al. [7], where it was observedthat 2-PTS suppresses, in a dose-dependent manner,the growth of HL-60 cells through the induction ofapoptosis initiated by oxidative stress. Although theremight be several mechanisms involved in the apoptosisof cancer cells, we believe that the effects of this sulfurcompound, and probably also of other OSCs, may belinked to mitochondrial expression levels and activityboth of rhodanese and of Trx in the cancer cells andthat the RNA interference technique could be used todemonstrate this hypothesis.Interestingly, high levels of Trx have also been associ-ated with cancer that is resistant to therapy, and lowTrx levels have been associated with apoptosis in gastriccarcinomas [62–64]. The Trx ⁄ Trd system is consideredto act as an endogenous antioxidant system in all livingcells, in addition to the glutathione system, so the mal-function of this antioxidant system in mitochondria canlead to an increase of intracellular ROS [65]. Mitochon-drial rhodanese–Trx ⁄ Trd-system oxidation by OSCscould lower the normal reducing activity of Trx and thusinhibit the activity of important enzymes, such as perox-iredoxin 3, an enzyme involved in H2O2metabolism inmitochondria, whose oxidation plays an important rolein the promotion of apoptosis [66].In conclusion, these studies contribute to extend theknowledge on the physiological role of rhodanese andmay represent a relevant starting point to elucidate theimplication of the rhodanese–Trx ⁄ Trd system in che-moprevention therapy approaches using sulfane sulfurcompounds, whose biochemical metabolism andcertain biological effects warrant further investigation.Materials and methods2-PTS synthesisSodium 2-propenyl thiosulfate was synthesized according toa method described by Chapelet et al. [67]. The productwas dried in vacuo , extracted with methanol and the extractwas purified by silica gel chromatography (methanol ⁄chloroform; 45 : 55, v ⁄ v). The purity and structure of thecompound were evaluated by RP-HPLC, LC-MS and1H-NMR.Cell proliferation assayHuT 78 human T-lymphoblastoid cells were purchasedfrom the ISS (Istituto Superiore di Sanita`, Rome, Italy).Approximately 0.2 · 106HuT 78 cells were pre-incubatedfor 24 h in RPMI 1640 (GIBCO, Milan, Italy) in the pres-ence of 1% glutamine, 10% heat-inactivated fetal bovineserum and antibiotics (1% penicillin and streptomycinsulfate) at 37 °C in air supplemented with 5% CO2andwere then exposed to 2-PTS for 24 and 48 h. The cells werecollected and counted, after staining with Trypan Blue(0.4% Tripan blu solution; Sigma-Aldrich, Milan, Italy), byoptical microscopy using a Thoma chamber. The rate ofgrowth inhibition by 2-PTS was calculated with respect tothe control culture taken as 100% growth.Cell cycle analysisThe cell cycle distribution of HuT 78 cells was measured byflow cytometry. Approximately 0.5 · 106harvested cellswere stained with 50 lgÆmL)1of propidium iodide (Sigma-Aldrich) in NaCl ⁄ Piwith 0.1% Triton X-100 and1mgÆmL)1of sodium citrate. Then, the cells were imme-diately analysed using a flow cytometer (FACSCalibur;Becton Dickinson, San Jose`, CA, USA) and the percentageof cells in each phase of the cell cycle was evaluated accord-ing to Nicoletti et al. [68].R. Sabelli et al. Sulfurtransferases and apoptotic natural sulfane sulfur compoundsFEBS Journal 275 (2008) 3884–3899 ª 2008 The Authors Journal compilation ª 2008 FEBS 3893[...]... performed using a Bruker win-nmr software package Protein extraction and western blot analysis Proteins were extracted from HuT 78 cells in 200 lL of 50 mm Tris–HCl, pH 7.09, containing a protease inhibitors cocktail (Sigma-Aldrich) and sonicated over four steps of 5 s and 1 min of pause in ice The samples were centrifuged (10 min, 5000 g, 4 °C) The protein contents were determined using the bicinchoninic... class of enzyme and of other proteins containing rhodanese domains for which biochemical characterization is still lacking A dysregulation of TST expression and activity induced by hydrogen sulfide (H2S) was observed, and this could result in the inability to detoxify effectively and be a factor in the cell loss and in ammation observed in ulcerative colitis and in colorectal cancer [16] Acknowledgements... during the organic synthesis of the 2-PTS compound, and Dr C Capo and Dr V Brunetti for technical support in some experimental studies We are grateful to Professor Silvia Pagani for kindly giving us the plasmid encoding the RhdA protein This work was supported, in part, by grants PRIN and FIRB of Italian MIUR References 1 Toohey JI (1986) Persulfide sulfur is a growth factor for cells defective in sulfur. .. The instrument was calibrated using myoglobin Proteolysis of RhdA-PS Limited proteolysis of RhdA was performed using 1% (w ⁄ w) trypsin (Sigma-Aldrich) in 50 mm Tris–HCl, pH 8, 0.3 m NaCl The reaction was stopped at different timepoints by the addition of SDS-PAGE sample buffer and boiling the samples for 2 min Subsequently, the samples were analysed using SDS-PAGE with a 15% resolving gel Thioredoxin... enzymes in Azotobacter vinelandii FEBS Lett 278, 151–154 Sulfurtransferases and apoptotic natural sulfane sulfur compounds 33 Pagani S, Sessa G, Sessa F & Colnaghi R (1993) Properties of Azotobacter vinelandii rhodanese Biochem Mol Biol Int 29, 595–604 34 Bordo D, Deriu D, Colnaghi R, Carpen A, Pagani S & Bolognesi M (2000) The crystal structure of a sulfurtransferase from Azotobacter vinelandii highlights... equilibria between intermediates in rhodanese catalysis J Biol Chem 258, 7894–7896 42 Melino S, Cicero DO, Forlani F, Pagani S & Paci M (2004) The N-terminal rhodanese domain from Azotobacter vinelandii has a stable and folded structure independently of the C-terminal domain FEBS Lett 577, 403–408 43 Horowitz PM & Criscimagna NL (1988) Sulfhydryldirected triggering of conformational changes in the enzyme... enzyme in 50 mm Tris–HCl buffer, pH 7.2 Kinetic analysis Rhodanese activity was measured by the discontinuous colorimetric assay described by Sorbo [71], where the pro¨ duction of thiocyanate from thiosulfate and cyanide was followed at 460 nm using a Perkin-Elmer spectrometer Briefly, 0.5 lg of enzyme (RhdA) was incubated in a reaction mixture containing 58 mm KCN and 58 mm sodium thiosulfate in 50... (2005) Growth inhibitory effect of alk(en)yl thiosulfates derived from onion and garlic in human immortalized and tumor cell lines Cancer Lett 223, 47–55 8 Westley B & Rochefort H (1980) A secreted glycoprotein induced by estrogen in human breast cancer cell lines Cell 20, 353–362 3896 R Sabelli et al 9 Hofmann K, Bucher P & Kajava AV (1998) A model of Cdc25 phosphatase catalytic domain and Cdk-interaction... Herman-Antosiewicz A, Marynowski SW & Singh SV (2006) c-Jun NH(2)-terminal kinase signaling axis regulates diallyl trisulfide-induced generation of reactive oxygen species and cell cycle arrest in human prostate cancer cells Cancer Res 66, 5379–5386 51 Hosono T, Fukao T, Ogihara J, Ito Y, Shiba H, Seki T & Ariga T (2005) Diallyl trisulfide suppresses the proliferation and induces apoptosis of human colon cancer... beta-tubulin J Biol Chem 280, 41487–41493 52 Wlodek L, Wrobel M & Czubak J (1993) Transamination and transsulphuration of L-cysteine in Ehrlich ascites tumor cells and mouse liver The nonenzymatic reaction of L-cysteine with pyruvate Int J Biochem 25, 107–112 53 Iciek MB, Rokita HB & Wlodek LB (2001) Effects of diallyl disulfide and other donors of sulfane sulfur on the proliferation of human hepatoma cell line . Rhodanese–thioredoxin system and allyl sulfur compounds Implications in apoptosis induction Renato Sabelli1, Egidio Iorio2, Angelo De Martino3,. 2B.Interaction of 2-PTS and allyl compounds withrhodanese In vitro interaction and kinetic studies were performedusing the recombinant Azotobacter vinelandii rhoda-nese
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