Biochemical Pharmacology 89 (2014) 210–216

Adenanthin targets proteins involved in the regulation of disulphidebonds

Angelika Muchowicz a, Małgorzata Firczuk a, Justyna Chlebowska a, Dominika Nowis a,b,Joanna Stachura a, Joanna Barankiewicz a, Anna Trzeciecka a, Szymon Kłossowski c,Ryszard Ostaszewski c, Radosław Zagozdzon a, Jian-Xin Pu d, Han-Dong Sun d,**,Jakub Golab a,e,*a Department of Immunology, Center of Biostructure Research, Medical University of Warsaw, Banacha 1A, 02-097 Warsaw, Polandb Genomic Medicine, Department of General, Transplant and Liver Surgery, Medical University of Warsaw, Banacha 1A, 02-097 Warsaw, Polandc Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Polandd Kunming Institute of Botany, Chinese Academy of Science, Kunming, Chinae Institute of Physical Chemistry, Polish Academy of Sciences, Department 3, Warsaw, Poland

A R T I C L E I N F O

Article history:

Received 28 December 2013

Received in revised form 26 February 2014

Accepted 27 February 2014

Available online 11 March 2014

Keywords:

Adenanthin

Cancer

Peroxiredoxin

Protein disulphide isomerase

Thioredoxin

A B S T R A C T

Adenanthin has been recently shown to inhibit the enzymatic activities of peroxiredoxins (Prdx) I and II

through its functional a,b-unsaturated ketone group serving as a Michael acceptor. A similar group is

found in SK053, a compound recently developed by our group to target the thioredoxin–thioredoxin

reductase (Trx–TrxR) system. This work provides evidence that next to Prdx I and II adenanthin targets

additional proteins including thioredoxin–thioredoxin reductase system as well as protein disulfide

isomerase (PDI) that contain a characteristic structural motif, referred to as a thioredoxin fold.

Adenanthin inhibits the activity of Trx-TR system and PDI in vitro in the insulin reduction assay and

decreases the activity of Trx in cultured cells. Moreover, we identified Trx-1 as an adenanthin binding

protein in cells incubated with biotinylated adenanthin as an affinity probe. The results of our studies

indicate that adenanthin is a mechanism-selective, rather than an enzyme-specific inhibitor of enzymes

containing readily accessible, nucleophilic cysteines. This observation might be of importance in

considering potential therapeutic applications of adenanthin to include a range of diseases, where

aberrant activity of Prdx, Trx–TrxR and PDI is involved in their pathogenesis.

� 2014 Elsevier Inc. All rights reserved.

Contents lists available at ScienceDirect

Biochemical Pharmacology

jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/bio c hem p har m

1. Introduction

Adenanthin, a diterpenoid isolated from the leaves of Isodon

adenanthus has been recently shown to inhibit the enzymaticactivities of peroxiredoxins (Prdx) I and II by targeting theirconserved resolving cysteines [1]. This mechanism of action seems

Abbreviations: DMSO, dimethyl sulfoxide; EAE, experimental autoimmune enceph-

alitis; FBS, fetal bovine serum; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-

trazolium bromide; PDI, protein disulphide isomerase; Prdx, peroxiredoxin; Trx,

thioredoxin; TrxR, thioredoxin reductase.

* Corresponding author at: Department of Immunology, Center of Biostructure

Research, The Medical University of Warsaw, 1a Banacha Str., F building, 02-097

Warsaw, Poland. Tel.: +48 22 599 2198; fax: +48 22 599 2194.** Corresponding author at: Institute of Botany, Chinese Academy of Sciences,

China. Tel.: +86 871 65223251.

E-mail addresses: hdsun@mail.kib.ac.cn (H.-D. Sun), jakub.golab@wum.edu.pl

(J. Golab).

http://dx.doi.org/10.1016/j.bcp.2014.02.022

0006-2952/� 2014 Elsevier Inc. All rights reserved.

to be essential for inducing differentiation of acute myeloidleukemia cells making this compound an interesting drugcandidate for further development in cancer treatment. Moreover,a recent study revealed that adenanthin is effective in prophylacticand therapeutic inhibition of MOG35–55-induced experimentalautoimmune encephalitis (EAE), an animal model of multiplesclerosis [2]. These potent immunomodulatory effects of ade-nanthin were associated with suppression of NF-kB signaling. Inthis work, we intended to provide further mechanistic insight intobiological actions of adenanthin.

Adenanthin contains a reactive functional group, an a,b-unsaturated ketone, which serves as a Michael acceptor. A similargroup is found in SK053 (19a in Ref. [3]), a compound recentlydeveloped by our group to target the thioredoxin–thioredoxinreductase (Trx–TrxR) system [3]. Importantly, Trx is one of themajor endogenous redox-regulating molecules with thiol-reduc-ing activity [4,5]. A DNA binding activity of NF-kB is stimulated by

A. Muchowicz et al. / Biochemical Pharmacology 89 (2014) 210–216 211

thioredoxin, which is involved in the reduction of its disulphidebonds [6]. Considering the structural similarities, involvement ofTrx in regulation of NF-kB binding to DNA, as well as the fact thatthe enzymatic reaction to measure Prdx activity is indirect becauseit involves an assay that couples its activity to the thioredoxinsystem comprised of NADPH, TrxR, and Trx [1], we decided toinvestigate whether adenanthin interferes with the enzymaticactivity of other enzymes containing a thioredoxin-fold, namelyTrx-TrxR system and protein disulfide isomerase (PDI).

Two-cysteine peroxiredoxins (Prdx) contain an active siteperoxidatic cysteine (CP) that undergoes oxidation by H2O2 tocysteine sulfenic acid (Cys-SOH) and a resolving cysteine (CR) thatreacts with the CP sulfenic acid to form a disulfide, further reducedby Trx [7]. Also, thioredoxin active site contains two cysteines:Cys32 and Cys35, which are responsible for protein disulfidereduction. The suggested mechanism of enzyme action is based onattack of nucleophilic Cys32 on a target protein disulfide to form acovalently linked intermediate. Subsequently, another nucleophil-ic attack of deprotonated Cys35 generates an oxidized form of Trx(Trx-S2) and reduces target protein [8]. Oxidized Trx is reduced byNADPH-dependent TrxR, and the enzyme is ready for anothercatalytic cycle. Due to the presence of highly nucleophilic cysteinesin the active site, a number of inhibitors of thioredoxin as well asother redox enzymes, contain an electrophilic center, whichcovalently interacts with cysteines [9].

2. Materials and methods

2.1. Extraction and isolation of adenanthin

The air-dried and powdered aerial parts of I. adenanthus (1.8 kg)were extracted three times with ethanol solution (3 � 7 L, each 3days) at room temperature and filtered. The filtrate was dried andpartitioned with ethyl acetate (Sigma, St. Louis, MO) (3 � 4 L). Theethyl acetate partition (110 g) was applied to silica gel (200–300 mesh) column chromatography, eluting with CH2Cl2–Me2CO(1:0–0:1 gradient system, Sigma), to give six fractions, which weredecolorized on MCI gel (Sigma), eluted with 90% MeOH–H2O(Sigma), to yield fractions A–F. Fraction B (250 g), brown gum, wassubjected to column chromatography over a silica gel (200–300 mesh) column, eluted with petroleum ether–Me2CO (1:0–0:1)gradient system (Sigma), to obtain five fractions, B1–B5. Ade-nanthin (16.5 g) was precipitated from fraction B2, it had a degreeof purity >98%. Adenanthin: mp: 253–255 8C; TLC (CHCl3:Me2CO,4:1 v/v): Rf = 0.62; [a]D

13=�76.0 (0.25 M in CHCl3); 1H NMR(500 MHz, CDCl3) d 5.77 and 5.17 (br s, each 1H), 5.51 (brs, 1H),5.26 (dd, J = 10.9, 3.0 Hz, 1H), 4.78 (br s, 1H), 3.66 (d, J = 2.6 Hz, 1H),3.33 (br s, 1H), 3.00 (br s, 1H), 2.54 (br s, 1H), 2.20, 2.00 and 1.76 (s,each 3H), 2.07 (m, 1H), 2.04 (d, J = 12.4 Hz, 1H), 1.93 (m, 1H), 1.84(overlapped, 1H), 1.84 (overlapped, 1H), 1.58 (d, J = 12.4 Hz, 1H),1.22 (s, 3H), 1.19 (s, 3H), 0.82 (s, 3H). 13C NMR (125 MHz, CDCl3) d205.7, 201.3, 170.4, 169.7, 169.2, 149.3, 113.9, 79.7, 78.5, 75.2, 68.3,54.3, 52.7, 50.5, 49.2, 37.8, 36.1, 35.7, 33.6, 31.5, 26.1, 22.2, 21.6,21.1, 21.0, 15.0; IR (Nujol): 3475, 1743, 1724, 1645, 1257, 1217,1030 cm�1; UV/Vis: lmax 231 nm; HRMS (m/z): [M]+ calcd. forHRMS (m/z): [M + Na]+ calcd. for C26H34O9Na, 513.2100; found,513.2106.

2.2. Cell lines and reagents

Acute promyelocytic leukemia (NB4) and human embryonickidney cell lines (HEK-293T) were obtained from DSMZ (Germany).Human Burkitt’s lymphoma (Raji) cell line was purchased from ATCC(Manassas, VA). Cells were cultured in RPMI-1640 mediumsupplemented with 5% (Raji) or 10% (NB4, HEK) heat-inactivatedfetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotic/

antimycotic solution (Sigma). The synthesis of SK053 [3] andadenanthin [1] was carried out according to previously describedmethods. Bacitracin was purchased from Sigma.

2.3. Measurement of thioredoxin activity

Human thioredoxin-1 was purified as described before [3]. Theactivity of the enzyme was confirmed in comparative experimentswith thioredoxin obtained from IMCO Corporation Ltd AB. Todetermine the influence of adenanthin on Trx�TrxR activity, theinsulin reduction assay was performed, as previously described [10].According to the protocol (IMCO Corporation Ltd AB, Stockholm,Sweden) the activity of recombinant Trx was measured in a finalvolume of 50 ml containing 50 mM Tris�HCl buffer (pH 7.6), 20 mMEDTA, 0.8 mM NADPH, 0.325 mM TrxR, 0.25 mM Trx, and 316 mMbovine insulin (all from Sigma) and the indicated concentrations ofinvestigated compounds. In all experiments at least seven differentconcentrations of inhibitors were used. The reduction of insulin byTrx was terminated after 30 min incubation in 37 8C by the additionof 8 mM DTNB [5,50-dithiobis-(2-nitrobenzoic acid] solution in 8 Mguanidine HCl (all from Sigma). The yellow pigment derived fromthionitrobenzoate was measured at 412 nm using an ELISA reader(Asys UVM 340 Microplate Reader, Biochrom, UK). The evaluation ofIC50 values was conducted with Four Parameter Logistic StandardCurves Analysis using SigmaPlot software. IC50 values are EC50� SD.

To evaluate Trx�TrxR activity in cells, NB4 and Raji cells weretreated with three different concentrations of inhibitors: 2, 4 and8 mM. Following 4 h of incubation cells were washed with PBS andlysed in 0.1% NP40 buffer (Sigma), supplemented with proteaseinhibitors cocktail (Roche Diagnostics, Basel, Switzerland). Aftermeasurement of protein concentration with BCA protein assay(Bio-Rad, CA) Trx�TrxR activity was determined according to themanufacturer’s protocol (IMCO Corporation Ltd AB). In eachsample 20 mg of total protein was used. Data points weremeasured in triplicates in individual experiments.

2.4. Time-dependent inhibition of recombinant Trx

To assess the nature of Trx inhibition by adenanthin, a modifiedinsulin reduction assay was used, where enzyme was pre-incubated with inhibitor before addition of other reactioncomponents. First, to obtain a reduced form of Trx, the enzymewas incubated with TrxR and NADPH for 20 min. Subsequently,adenanthin at 1, 2, 4, 6, or 12 mM concentration was added for 30,60, 120 and 180 min of incubation. The insulin reduction assayrequires the excess of TrxR. Since the inhibition of TrxR activity byadenanthin cannot be excluded, the additional amount of thisenzyme was added to the reaction. This step allows to minimizethe possible influence of the adenanthin on TrxR activity.Additionally, in order to investigate whether the inhibition isirreversible, the reaction was 5-fold diluted (from 20 ml to 100 mlfinal volume) and initiated with insulin. The final concentrations ofreaction components were as follows: 0.25 mM Trx, 6.5 mM TrxR,1 mM NADPH, and 0.2–2.4 mM adenanthin. Reaction was carriedout for 30 min in 37 8C and was terminated as described above. Theresults represent means from two independent experiments.

2.5. Expression and purification of human PDI

Human PDI cDNA lacking a 17 amino acid long signaling sequence(PDI18–508) was amplified from human Burkitt’s lymphoma Raji cells(CCL-86, ATCC, Manassas, VA) using the following primers: forward:50-GGCCGGTACCGGACGCCCCCGAGGAGG-30 and reverse: 50-GGCGAAGCTTTTACAGTTCATCTTTCACAGCTTTCTGG-30 and clonedinto pETMM11 vector (a kind gift of G. Stier and D. Drechsel, MPI,Dresden) resulting in addition of N-terminal His-tag. Protein

A. Muchowicz et al. / Biochemical Pharmacology 89 (2014) 210–216212

expression was carried out in Escherichia coli strain BL21(DE3) grownin LB medium (Sigma) at 30 8C, and induced at an OD600 of 0.8 for 24 hwith 1 mM IPTG (isopropyl b-D-1-thiogalactopyranoside, Fluka,Milwaukee, MI). Bacterial cells were pelleted by centrifugation(3000 � g for 15 min) and the pellet was resuspended in 20 ml oflysis buffer composed of CelLytic B, 20 mM Tris pH 7.5, 300 mM NaCl;DNAse 20 mg/ml; lysozyme 0.2 mg/ml (all from Sigma) and lysed for15 min at RT. The cell debris was removed by centrifugation(15,000 � g for 60 min). The clarified supernatant was filteredthrough a 0.45 mm PES syringe filter (Merck Millipore, Billerica, MA)and loaded onto the HiTrap FF crude 5-ml Ni Sepharose column (GEHealthcare Life Sciences AB, Uppsala, Sweden) equilibrated with20 mM Tris pH 7.5, 150 mM NaCl, 5 mM imidazole buffer. The nickelaffinity chromatography and subsequent gel filtration were per-formed using Akta Avant 25 operated by Unicorn 6 software (GEHealthcare Life Sciences, Upsala, Sweden). The protein was elutedusing 20 mM Tris pH 7.5, 150 mM NaCl buffer with imidazolegradient varying from 5 to 300 mM. Next, the protein was furtherpurified with gel filtration chromatography using Superdex 200 10/300 GL column followed by HiLoad 16/600 Superdex 75 pg column(both from GE Healthcare Life Sciences) equilibrated with 20 mMTris, 300 mM NaCl buffer. The eluted fractions containing PDI wereconcentrated using VivaSpin 10 kDa concentrator (Sartorius), mixed1:1 with glycerol and stored aliquoted at �20 8C. The purity ofrecombinant human PDI was verified in SDS-PAGE with subsequentCoomasie blue (Sigma) staining.

2.6. PDI activity assay

PDI activity was assessed with a turbidimetric assay of insulindisulfide reduction. To investigate the influence of tested com-pounds (adenanthin and bacitracin) on recombinant human PDI, theprotein (1.7 mM) was incubated with indicated concentration of thecompounds at 37 8C for 1 h in the reaction buffer (100 mM sodiumphosphate, 2 mM EDTA, pH 7.4). Next, the reaction mixtureconsisting of DTT (270 mM, Fluka) and bovine insulin (545 mM,Sigma) was added. The reduction reaction was catalyzed by PDI atroom temperature, and the resulting aggregation of reduced insulinchains was measured at 650 nm using Asys UV340M microplatereader. The IC50 was estimated after 30 min of reaction with FourParameter Logistic Standard Curves Analysis using SigmaPlotsoftware. IC50 values are EC50� SD.

2.7. Cytostatic/cytotoxic assay

The cytostatic/cytotoxic effects were measured with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) as-say (Sigma). Tumor cells were dispensed into 96-well plates andincubated for 24 h with investigated compounds at indicatedconcentrations. Subsequently MTT solution in a concentration of5 mg/ml was added to each well of plate. After 4 h, plates werecentrifuged, and cells were lysed with DMSO. Finally, the absorbancewas measured at 570 nm using Asys UV340M microplate reader.

2.8. Affinity purification of adenanthin-binding proteins

Incubation of 1 � 107 of Raji and NB4 cells with 50 mM biotin-tagged adenanthin or free biotin was carried out in serum freeRPMI medium at 37 8C. After 4 h cells were harvested, washedtwice with PBS, resuspended in a buffer containing 1% NP40 in PBSand frozen at �70 8C. For lysis, cells were thawed and incubated onice for 30 min with frequent, vigorous mixing. Next, the sampleswere centrifuged 30 min, 20,000 x g, at 4 8C, the supernatants werecollected and total protein concentration was measured withBradford method (Bio-Rad). For each experimental group, 0.5 mgof total protein was incubated for 1 h with monomeric neutravidin

beads pre-treated according to manufacturer’s procedure (ThermoScientific). After four extensive washes with buffer containing20 mM Tris pH 8.8, 0.5% NP-40, 500 mM NaCl alternating withdistilled water, specifically bound proteins were eluted with 5 mMbiotin in PBS. In order to investigate the level of selected proteins,the eluates were subjected to Western blotting, using specific anti-Prdx-1 (Sigma Aldrich, HPA007730), anti-Trx-1 (Cell Signaling,anti-Trx-1, 2429), anti-Tubulin (Calbiochem, CP06), anti-Actin(Sigma Aldrich, A3854) antibodies.

2.9. Assessment of glutathione levels

Raji cells were seeded at 2 � 105/well in a 24-well plate andincubated with adenanthin at indicated concentrations in the cellculture medium for 2 h in 37 8C in 3 independent repeats.Subsequently, cells were centrifuged, washed with PBS andresuspended in 500 ml of PBS. The GSH/GSSG-GloTM Assay (Promega,V6611) was performed according to manufacturer’s protocol using25 ml of each cell suspension (1 � 104 cells). Thiol-reactive agent(10 mM N-ethylmaleimide) was used to non-specifically reduceGSH levels. Vehicle control (cells incubated with DMSO) and no-cell control (PBS) were also included. The glutathione concentra-tions [mM] were determined in final reaction volume (200 ml)using GSH and GSSG standard curves and the ratio of GSH/GSSGwas calculated by (GSH-2 � GSSG)/GSSG equation.

2.10. Lentiviral vector production and transduction of target cells

Gene encoding human Trx-1 was PCR-cloned from cDNAobtained from Raji cells line and inserted into the multicloning siteof the puromycin-resistant mammalian expression vector pLVX-IRES-Puro (Clontech). Expressing vector as well as packing andenvelope plasmids were introduced into HEK cells by means ofGenJuice (Novagene, EMD4Biosciences Inc., Gibbstown, NJ).pMD2.G and psPAX2 plasmids were generous gifts from prof.Didier Trono (Ecole polytechnique federale de Lausanne,Switzerland). Subsequently lentiviral particles were used for Rajicell transduction according to the protocol described before [11]. Inorder to obtain Trx-1 knock-down Raji cells, lentiviral particlescontaining proper shRNA sequences were used (MISSION1

pLKO.1-puro vector, Sigma) according to the manufacturer’sprotocol. Control cells were transduced with MISSION1 pLKO.1-puro non-mammalian shRNA control transduction particles, whichtargets no known mammalian genes. Stable transduced cell lineswere established for each shRNA-containing-vectors by puromycinselection and mixtures of clones were used in the experiments. Thelevels of Trx-1 were measured with Western blotting.

2.11. Statistics

Data were calculated using Microsoft Excel 2007, SigmaPlot 8.0and/or GraphPad Prism 6. In all experiments the differences forsignificance were analyzed by non-parametric Mann–Whitnney U

test.

3. Results

SK053 (compound 19a in [3]) is a b-acyloxyacrylamide with theelectrophilic center that acts as an irreversible inhibitor of Trx/TrxRsystem. An analogous electrophilic center can be found in thechemical structure of adenanthin (Fig. 1), a novel compoundidentified recently to inhibit Prdx I/II enzymes [1]. Hence, theinsulin reduction assay was used in this work to determine theinfluence of adenanthin on recombinant thioredoxin–thioredoxinreductase activity. The reaction was carried out for 30 min at 37 8C.

Fig. 1. Chemical structure and mechanisms of action of SK053 and adenanthin. SK053 and adenanthin contain an activated double bond that serves as a Michael acceptor (red),

which can react with ‘‘thioredoxin-like’’ proteins in a mechanism that involves cysteine alkylation. (For interpretation of the references to color in this figure legend, the

reader is referred to the web version of this article.)

A. Muchowicz et al. / Biochemical Pharmacology 89 (2014) 210–216 213

The calculated IC50 values are 10.9 � 1.6 mM for adenanthin, and3.0 � 0.2 mM for SK053, as reported before [3].

To investigate if adenanthin irreversibly inhibits Trx, thereaction with recombinant Trx-1 and adenanthin pre-incubationwas carried out. Enzymatically reduced Trx was incubated withdifferent concentrations of adenanthin for 30, 60, 120 and 180 min.Subsequently, the reagents were five-fold diluted by the additionof insulin, excess concentration of TrxR and NADPH, and incubated

Fig. 2. Adenanthin inhibits the activity of Trx. (A) The time depended inhibition of recombi

thioredoxins was determined in Raji and NB4 cells, incubated with SK053 or adenanthin

after 24 h of incubation with MTT assay. * P < 0.05 Mann–Whitnney U test.

at 37 8C for the next 30 min. The dilution of the reagents allows toinvestigate whether the reaction of inhibitor binding to theenzyme is irreversible. As shown in Fig. 2A, a decrease inenzymatic activity depends on the pre-incubation time andconcentration of the inhibitor, with an almost complete inhibitionobserved after 180 min of pre-incubation with adenanthin. Theseresults indicate that adenanthin is a potent and irreversibleinhibitor of thioredoxin.

nant Trx was evaluated with insulin reduction assay. (B) The activity of intracellular

for 4 h. (c) The cytostatic/cytotoxic effects of SK053 or adenanthin were evaluated

Fig. 3. Adenanthin binds to Trx. Raji and NB4 cells were incubated with 50 mM of

biotin-tagged adenanthin (Ade-Bio) or free biotin (Bio) and lysed. Biotinylated

proteins were precipitated from 0.5 mg of total protein using monomeric avidin

agarose beads, eluted with 5 mM biotin in PBS and the levels of Prdx-1 and Trx-1

were detected with Western blotting.

A. Muchowicz et al. / Biochemical Pharmacology 89 (2014) 210–216214

Next, the potential of adenanthin to inhibit Trx intracellularlywas investigated in two different cell lines: Raji and NB4. Theresults for both cell lines are similar and indicate that thiscompound decreases the activity of Trx in cells after 4 h ofincubation, but to a slightly lower extent as compared with SK053(Fig. 2B). Nevertheless, in a cell viability assay adenanthin exertsmore potent cytostatic/cytotoxic effects against tumor cells, ascompared with SK053 (Fig. 2C).

To see whether adenanthin covalently binds to Trx in cells weused biotin-tagged adenanthin. Equal amounts of lysates obtainedfrom Raji and NB4 cells pre-incubated with biotin-adenanthin orfree biotin as a control underwent neutravidin affinity chroma-tography and the eluted proteins were analyzed with Westernblotting (Fig. 3). Apart from the previously identified Prdx-1, Trx-1was also detected as adenanthin-binding protein in both cell lines.Neither tubulin nor actin could be detected, suggesting thatadenanthin binds only selected proteins with nucleophiliccysteines.

To further investigate adenanthin specificity, we measuredlevels of reduced and oxidized glutathione (GSH and GSSG,respectively) in Raji cells after incubation with adenanthin. Bothadenanthin and a thiol-reactive N-ethylmaleimide (NEM) de-creased GSH/GSSG ratio in Raji cells (Fig. 4A). GSH and total

Fig. 4. Adenanthin does not deplete glutathione levels. (A) Reduced glutathione (GSH) to ox

GSSG were determined using the GSH/GSSG-GloTM Assay (Promega, V6611). 10 mM N

represent the mean � standard deviation (n = 3).

glutathione were significantly reduced upon NEM treatment andonly slightly reduced upon the highest, 8 mM adenanthin (Fig. 4Band C). Only in adenanthin treated cells, a drop in GSH wasaccompanied by an increase in GSSG (Fig. 4D). The above resultsconfirm that adenanthin triggers oxidative stress and exclude thatit is a non-specific thiol-depleting agent.

To determine whether Trx-1 levels can influence the cytostatic/cytotoxic effects of adenanthin, mixtures of clones of Raji cells witheither up-regulated (Raji-pLVX-Trx1) or silenced (Raji-shRNA-Trx1) thioredoxin were incubated with various concentrations ofadenanthin. As shown in Fig. 5A, Trx activity was significantlyincreased in Raji cells overexpressing Trx-1. After incubation ofthese cells with adenanthin, the activity decreased almost to thecontrol level (Fig. 5A, upper panel). On the other hand, Trx activitywas strongly decreased in Trx-1 knock-down cells and incubationof these cells with adenanthin only minimally reduced the activity.Overexpression of Trx-1 rendered tumor cells more resistant tocytostatic/cytotoxic effects of adenanthin (Fig. 5B, upper panel),while knock-down of this enzyme increased the cells sensitivity tothe investigated inhibitor (Fig. 5B, bottom panel). These resultsindicate that sensitivity to adenanthin correlates with Trx-1 levelsexpressed in cells. Moreover, Trx-1 expressed in Raji cells can beintracellularly inhibited by adenanthin.

Dozens of thioredoxin-fold containing enzymes are expressedin cells [12]. They are important for the maintenance of cellularredox homeostasis and increasing amount of data reports theirimportance in supporting cancer cell growth [13,14]. To investi-gate the influence of adenanthin on the activity of protein disulfideisomerase (PDI), another enzyme containing a thioredoxin fold, weemployed a turbidimetric assay of insulin disulfide reduction. Asshown in Fig. 6, adenanthin inhibits the activity of PDI with IC50

value 7.3 � 0.3 mM.

4. Discussion

Cellular proteins undergo a variety of post-translationalchemical modifications. These include (i) changes to amino acid

idized glutathione (GSSG) ratio, (B) GSH, (C) total glutathione (GSH + GSSG) and (D)

-ethylmaleimide (NEM) was used as a non-specific, thiol-reactive agent. Graphs

Fig. 5. Effects of Trx-1 levels on the cytostatic/cytotoxic activity of adenanthin. (A) The activity of Trx evaluated in Raji cells with different expression levels of the enzyme, after 4 h

of incubation with adenanthin. (B) Cytostatic/cytotoxic effects of adenanthin measured in Trx-1 overexpressing cells (Raji-pLVX-Trx) and Trx-1 knock-down cells (Raji-

shRNA-Trx) measured with MTT assay. Control cells were either mock-transfected with empty lentivirus (pLVX) or cells transduced with a non-tageting shRNA (NT-shRNA). *

P < 0.05 Mann–Whitnney U test. (C) The protein levels were examined with immunoblotting. Densitometry was measured and calculated by means of AIDA software

(Raytest, Straubenhardt, Germany). Data show mean values from two independent repeats � S.D.

A. Muchowicz et al. / Biochemical Pharmacology 89 (2014) 210–216 215

side chains (such as addition of phosphate, acetyl, methyl orprenyl groups), (ii) changes in peptide bonds resulting in cis–trans isomerization, and (iii) changes in disulfide bonds allowingfor folding and conformational reconfigurations of matureproteins [15]. All these modifications are highly dynamic andregulated in a number of enzymatic reactions. Disulfide bondslink pairs of cysteines within or between proteins. While themajority of such bonds are relatively stable throughout theprotein lifetime, some can be reductively cleaved, usually in aregulated way, and are referred to as allosteric disulfide bonds[16]. These reactions, catalyzed by oxidoreductases or by thiol-disulfide exchanges, participate in the control of protein function

Fig. 6. Adenanthin inhibits the activity of PDI. PDI activity was assessed with a

turbidimetric assay of insulin disulfide reduction.

[17]. Among the oxidoreductases are enzymes participating inred-ox reactions, protein folding or antigen degradation, such asthioredoxin, protein disulfide isomerase family or gamma-interferon-inducible lysosomal thiol reductase. Although thenumber of proteins with allosteric disulfide bonds is relativelylow (only 27 are well characterized so far [15]), their significancein the regulation of cancer-related proteins function is increas-ingly being recognized. Therefore, regulation of allosteric bondformation becomes a potential target for novel anticancertherapeutics.

In this work we demonstrated that adenanthin inhibited theactivity of two human enzymes which belong to thioredoxinfamily and are involved in allosteric disulfide bond formation: Trx-1 and PDI (Figs. 2 and 6). The IC50 for inhibition of Trx-TR systemand for PDI were 10 mM, and 7.3 mM, respectively, which iscomparable to other known inhibitors of theses enzymes. We showthat adenanthin is covalently bound to Trx-1 in Raji cells (Fig. 3)and that Trx activity was significantly diminished in cells pre-incubated with adenanthin (Fig. 2). These results indicate thatadenanthin binds Trx-1 and inhibits its activity in cells. Moreover,we demonstrated that the expression level of Trx-1 influenced thesensitivity to adenanthin (Fig. 5).

In conclusion, the results of our studies indicate thatadenanthin is a mechanism-based, rather than an affinity-basedinhibitor of enzymes containing nucleophilic active site cysteines,including those involved in the regulation of allosteric disulfidebonds formation. Next to Prdx I and II, it also targets Trx andprobably also PDI. Importantly, the more promiscuous activity of

A. Muchowicz et al. / Biochemical Pharmacology 89 (2014) 210–216216

adenanthin may affect its potential therapeutic applications. Forexample, the HIV gp120 can be reduced by PDI, Trx orglutaredoxin-1 allowing for virus entry into cells [18]. Otherproteins containing allosteric disulfides include CD4, MICA,common g chain of IL-2 receptor, VEGFC (vascular endothelialgrowth factor C), VEGFD, CSK (C-terminal SRC kinase), tissue factor,plasmin and many others [16]. Therefore, compounds such asadenanthin or SK053 can be considered as potential therapeutics inthe management of a range of diseases, where aberrant activity ofthese proteins is involved in their pathogenesis.

Acknowledgments

We would like to thank Anna Czerepinska and Elzbieta Gutowskafor technical assistance. A.M. is recipient of the Polish L’Oreal-UNESCO Awards for Women in Science. This work was supported inpart by grants NN405127640 (A.M.), IP2011 012671 (M.F.), IP2012048172 (D.N.) and IP2011 038971 (D.N.) from the Ministry of Scienceand Higher Education, Medical University of Warsaw Grant 1M19/PM13D/12/12 (A.M), grant no 2012/07/B/NZ7/04183 from theNational Science Centre (R.Z.), grant no 21322204 from the NationalNatural Science Foundation of China (H-D.S.) and by a grant from theEuropean Commission 7th Framework Programme: FP7-REGPOT-2012-CT2012-316254-BASTION (J.G.).

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