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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, United Kingdom and 2 Science Applications International Co.-Frederick, Inc., Screening Technologies Branch, Laboratory of Functional Genomics, National Cancer Institute, Frederick, Maryland
Requests for reprints: Tracey D. Bradshaw, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, NG7 2RD, United Kingdom. Phone: 44-115-951-3419; Fax: 44-115-951-3412; E-mail: tracey.bradshaw{at}nottingham.ac.uk.
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Abstract |
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10,000 cancer-related genes, expression of thioredoxin reductase was up-regulated >3-fold. Furthermore, quinols 1 and 2 inhibited insulin reduction, catalyzed by thioredoxin/thioredoxin reductase signaling in a dose-dependent manner (IC50 < 6 µmol/L). Results are consistent with a mechanism of action of novel antitumor quinols involving inhibition of the small redox protein thioredoxin. |
Introduction |
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In our laboratory, two lead molecules have emerged (1 and 2) from the heteroaromatic cyclohexadienones (quinols) program, which elicit potent antitumor activity against HCT 116 and HT29 colon-derived, and MCF-7 and MDA 468 breast-derived human carcinoma cell lines. However, when the intriguing profile of tumor cell growth inhibition and cytotoxicity was initially uncovered, a molecular target was elusive: these agents exacted antitumor activity via unknown mechanism(s) of action.
In this article, we describe the multidisciplinary approach undertaken to elucidate mechanisms involved in the antitumor activity of novel quinols, exposing thioredoxin as a molecular target. The implications of these findings are discussed.
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Materials and Methods |
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Mammary carcinoma [estrogen receptor–positive (ER+) MCF-7 and estrogen receptor–negative (ER–) MDA-468] and colon carcinoma (HCT 116 and HT29) cell lines were subcultivated twice weekly in RPMI 1640 supplemented with 10% fetal bovine serum and incubated at 37°C in an atmosphere of 95% air, 5% carbon dioxide. To minimize phenotypic drift, cells were maintained in culture for 4 months before being discarded and early passage cells resurrected from liquid nitrogen storage.
National Cancer Institute In vitro Cytotoxicity Assays
Cell culture and drug application procedures have been described previously (3). Briefly, cell lines were inoculated into a series of 96-well microtiter plates, with varied seeding densities depending on growth characteristics of each cell line. Following a 24-hour drug-free incubation, test agents were added at five 10-fold dilutions with a maximum concentration of 100 µmol/L. Cellular protein levels were determined after 48 hours of drug exposure by sulforhodamine B colorimetry.
Growth Inhibition
Cells were seeded into 96-well microtiter plates at a density of 5 x 103 per well and allowed 24 hours to adhere before drugs were introduced (final concentration, 0.1 nmol/L to 100 µmol/L; n = 8). Serial drug dilutions were prepared in medium immediately before each assay. Viable cell masses at the time of drug addition (time 0), and following 72 hours of drug exposure were determined by cell-mediated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. MTT was added to each well (final concentration, 400 µg/mL) and plates incubated at 37°C for 4 hours to allow reduction of MTT by viable cell dehydrogenases to an insoluble formazan product. Well supernatants were aspirated and cellular formazan solubilized by addition of DMSO/glycine buffer (pH 10.5; 4:1). Cell growth as well as drug activity were determined by measuring absorbance at 550 nm using an Anthos Labtec systems plate reader.
Cell Cycle Analysis
Cell cycle distributions were analyzed following exposure of cells to compounds 1 (1 µmol/L) and 2 (0.5 µmol/L) by flow cytometric analysis of cellular DNA content (4). Briefly, culture medium including detached cells was collected and the attached cells trypsinized. Attached and floating cells were pooled, pelleted, and washed twice in ice-cold PBS. Cells were then resuspended in 0.5 mL fluorochrome solution containing 50 µg/mL propidium iodide, 0.1% sodium citrate, 0.1% Triton X-100, and 0.1 mg/mL RNase A. Following 1 hour incubation at 4°C protected from light, the cells were analyzed on a Beckman Coulter EPICS-XL flow cytometer. Data analysis was carried out using EXPO32 software.
Gene Array
Preparation of RNA. Cells were seeded into 160-cm3 flasks at appropriate densities and allowed 48 hours to attach and reach logarithmic growth. Medium was replenished and drug introduced before incubation of cells for a further 24 hours. Cells were harvested at 
75% confluency, washed in ice-cold PBS, pelleted, and counted. Tri reagent (Sigma, Poole, United Kingdom) was added to the cell pellets (1 mL/1 x 107 cells) and samples were vortexed and incubated at room temperature for 5 minutes. Chloroform was added to each sample (0.2 mL/1 x 107 cells) before vigorous shaking (20 seconds). Samples were incubated at room temperature for a further 3 minutes before centrifugation at 12,000 x g, 15 minutes at 4°C. The clear upper phase was transferred to a new tube and isopropanol (0.5 mL/1 x 107 cells) was added; samples were vortexed and again centrifuged at 12,000 x g for 15 minutes at 4°C. The supernatant was removed, and the RNA pellets washed with 75% ethanol. After centrifugation (7,500 x g, 5 minutes, 4°C), ethanol was removed and RNA pellets air-dried. RNA was resuspended in RNase-free distilled H2O and quantitated at 
260 nm. A ratio of 260/280 > 1.7 ensured pure RNA, which was immediately frozen to avoid degradation.
Microarray analyses. RNA samples extracted from untreated HCT 116 cells and cells exposed to 1 and 10 µmol/L 1 for 24 hours were competitively hybridized against a cDNA array containing 10,000 elements. The RNA from each drug-treated sample was reverse transcribed and an amino-allyl modified dUTP incorporated which binds the CY3 fluorescent dye. Control, untreated samples were modified in the same manner but with CY5 dye bound to cDNA (reverse dye incorporation). Equal amounts of the two samples were hybridized against five cDNA arrays (Advanced Technology Center, Center for Cancer Research, NCI). The ability of 2 (0.5 and 1 µmol/L) to alter gene expression in HCT 116 colon carcinoma cells was determined after 24 hours of exposure. RNA was isolated as described and gene expression evaluated by competitive hybridization of control versus drug-treated cDNA samples as described above, on triplicate microarrays containing 20,000 oligonucleotides (Advanced Technology Center, Center for Cancer Research, NCI). All data was analyzed through the Computer Information Technology Center's mAdb web site.4 Gene lists generated were subjected to DAVID and EASE bioinformatic tools developed by the Laboratory of Immunopathogenesis and Bioinformatics at SAIC, Frederick for the National Institute of Allergy and Infectious Diseases of the NIH. EASE determined GO functional classifications where the abundance of genes in the selected set (up-regulated or down-regulated) was significantly higher (Bonferroni corrected P < 0.01) than that in the Locus Link database, suggesting functional classifications of identified genes, which did not represent random selection.
Western Blot
Whole cell lysates were prepared for examination of protein expression from untreated cultures and following exposure of cells to compounds 1 and 2. Following protein determination (n = 3; ref. 5) and addition of sample buffer, samples were heated to 95°C for 5 minutes and solubilized proteins (50 µg) were separated by SDS-PAGE (10%). Proteins were electroblotted to polyvinylidene difluoride membranes and probed using an anti-CDK-1 primary antibody (Oncogene Research Products, San Diego, CA) and a secondary antibody conjugated to horseradish peroxidase (Pierce, Rockford, IL). Membranes were then treated with SuperSignal West Pico enzyme chemiluminescence substrate (Pierce) before exposure to X-ray film (Fuji, Tokyo, Japan) for 5 minutes. Membranes were stripped using stripping buffer [0.15 mol/L glycine and 0.4% SDS (pH 2.5)], washed, and blocked before being reprobed with antibodies against thioredoxin (Becton Dickinson, Franklin Lakes, NJ), thioredoxin reductase (TR; Lab Frontier, Seoul, Korea) and actin (Oncogene Research Products) using a similar detection system. Actin levels were used to verify protein loading. Three separate sets of samples were analyzed for all four proteins. Molecular weight markers were included in all blots to confirm detection of proteins of the correct molecular weight.
Mass Spectrometry
Human thioredoxin protein (Imco, Stockholm, Sweden) was dissolved (1 mg/mL, 86 µmol/L) in 100 mmol/L ammonium bicarbonate (pH 8.1)/1 mmol/L tris(2-carboxyethyl)phosphine (TCEP). Compound 1, final concentration 1 mmol/L (5% DMSO), or vehicle alone was added. After incubation (15 minutes, 37°C), 5-µL aliquots were partially purified by washing (0.1% formic acid/water on a C-18 reversed-phase ZipTip; Millipore, Billerica, MA) before elution in 50% acetonitrile in 0.1% formic acid/water and analysis on a Q-Tof-2 mass spectrometer (Micromass, Manchester, United Kingdom). Remaining samples were trypsin-digested (Promega, San Luis Obispo, CA) overnight using a 1:6 ratio of trypsin to thioredoxin. Trypsin, a stable and aggressive protease, specifically cleaves proteins on the COOH-terminal side of arginine (R) and lysine (K) residues. Digestion was stopped by the addition of 0.1% formic acid and samples reanalyzed by mass spectrometry and the peptide mass fingerprints compared. Modified peptides were selected for fragmentation in tandem mass spectrometry (MS/MS) mode to identify point(s) of interaction. Selected ions were fragmented using, typically, a range of applied voltages of 25 to 35 eV in the presence of argon gas. Resultant daughter ion spectra were deconvoluted using MaxEnt3 software and subsequently applied to the sequencing portion of BioLynx software included in the MassLynx data analysis platform (Waters, Hertsfordshire, United Kingdom) for sequence identification and annotation.
Insulin Reduction Assay
Microtiter plate colorimetric assays, based on the increase in absorbance at 405 nm, which occurs when dithionitrobenzoic acid (DTNB) is reduced by the enzyme-mediated transfer of reducing equivalents from NADPH, were done for TR/thioredoxin–dependent insulin reduction (6). TR/thioredoxin–dependent insulin reducing activity was measured in incubates (final volume, 100 µL) containing 100 mmol/L HEPES buffer (pH 7.2), 5 mmol/L EDTA (HE buffer), 1 mmol/L NADPH, 1 µmol/L TR, 0.8 µmol/L thioredoxin, and 2.5 mg/mL bovine insulin. Incubations were for 30 minutes at 37°C in flat-bottomed 96-well microtiter plates. The reaction was stopped by the addition of 125 µL of 6 mol/L guanidine-HCl, 50 mmol/L Tris (pH 8.0), and 10 mmol/L DTNB and absorbances were measured at 405 nm. Appropriate control samples were included in the assay in which TR, thioredoxin, or insulin were omitted from incubates. To determine the capacity of 1 and 2 to inhibit insulin reduction by TR/thioredoxin signal transduction, quinols (10 µL) were included in incubation mixtures. Insulin reduction assays were done in which all reagents were introduced simultaneously. In addition, to elucidate further the inhibitory nature of TR/thioredoxin signal transduction by 1 and 2, reagents excluding insulin were incubated (30 minutes, 37°C) before introduction of insulin. There followed a further 30 minutes incubation at 37°C before the reaction was stopped as described above and absorbance read at 405 nm.
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Results |
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Cell cycle analyses. MCF-7, MDA 468, HCT 116, and HT29 cells, challenged with growth inhibitory concentrations of 1 and 2, show immediate onset of profound G2-M cell cycle block, at the expense of G1 and initially, S-phase cells. Representative cell cycle perturbations are illustrated in Fig. 3, following treatment of HCT 116 cells with 1 µmol/L 1 and 500 nmol/L 2. As treatment times extended beyond 16 hours, numbers of cells within S phase began to increase once more, and a pre-G1 apoptotic peak became evident; however, no significant release of cells blocked within the G2-M cell cycle phases was evident.
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4-fold increase or decrease in gene expression following drug treatment in more than two of the replicate assays. Intriguingly, the only gene whose transcription was induced by 1 µmol/L 1 and met these criteria was TR. All other selected genes whose transcription was altered following exposure of cells to 1 µmol/L 1 were down-regulated, although after 10 µmol/L treatment, the zinc finger protein 177 (ZNF177) and the basic transcription element binding protein 1 (BTEB) genes showed increased expression. As both these genes are potential transcription regulators, the higher concentration may regulate gene transcription by additional mechanisms. This hypothesis is supported by the observation that following treatment of HCT 116 cells with 10 µmol/L 1, and using the same selection criteria, transcription of 32 genes was up regulated and 11 genes down-regulated (data not shown). Among the former, TR was modestly induced (2- to 3-fold; Fig. 4A). The most highly induced genes encoded heat shock proteins, and transcription factors that tended to be stress response genes. Furthermore, transcription of GADD45, the DNA damage response gene was enhanced, as was Il-8, another common stress response gene. The most highly down-regulated gene was that encoding CDK-1, the kinase responsible for cycling the cell through G2-M. A further interesting observation was that asparagine synthetase transcription was also down-regulated in response to both 1 and 10 µmol/L 1.
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4-fold increase or decrease in expression in 5 of 6 arrays, of the 10 genes that met these criteria (Fig. 4B), transcription of TR was the most significantly induced gene. Heme oxygenase 1 (HO-1) was also induced dose-dependently.
When less stringent criteria were adopted (1.7-fold induction of gene expression), transcription of 148 genes was up-regulated. When these data were subjected to EASE functional classification, genes clustered within membranous compartments, such as endoplasmic reticulum and lysosomes were observed, including an endoplasmic reticulum thioredoxin superfamily member (molecular weight 18 kDa).
EASE evaluation established that down-regulated genes meeting the same criteria, fell into categories of cell surface receptor linked signal transduction, G-protein coupled receptor signaling, signal transducer activity, and cell communication, indicating overall inhibition of signal transduction.
Protein expression. Representative Western blots, following detection of thioredoxin, CDK1, TR, and loading control actin in lysates of HCT 116 cells are shown in Fig. 5. Cells were exposed either to 1, 2 (100, 500 nmol/L, and 1 µmol/L), or DMSO vehicle for 24 hours before preparation of cell lysates. Dose-dependent down-regulation of CDK1 protein was detected following exposure of cells to both quinol analogues, concordant with the observed cell cycle block and reduced cdk1 gene transcription following treatment of HCT 116 cells with 1 µmol/L 1. In contrast, compounds 1 and 2 evoked increased expression of TR protein, in a dose-dependent manner, again corroborating microarray data. There was no significant alteration in levels of thioredoxin detected in lysates of cells treated with either analogue. Protein content introduced into gel wells remained constant (50 µg) a fact confirmed by detection of actin in each lysate sample.
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0.03% and 0.04% from the calculated expected mass, thought to be caused by reduced quality of spectra upon incubation with drug, as drug-bound protein did not ionize or "fly" as well in the mass spectrometer as the control incubated thioredoxin. A peak corresponding to nonmodified thioredoxin was not present after drug incubation, suggesting that all the protein had bound drug.
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A 2+ ion of mass/charge 732, which would correspond to the expected peptide fragment constituted by residues 37-48 (expected mass 1463), a region incorporating no Cys residues, was detected in both the control and drug-incubated protein samples.
The peptide corresponding to residues 49 to 72 (incorporating 2 Cys residues), expected mass 2,719, was also detected as a 3+ ion of mass/charge 907. The drug-incubated protein did not confer this ion but a 3+ ion of mass/charge 988, which would correspond to the peptide fragment of residues 48 to 71 plus 1 (only) molecule of 1 (expected calculated mass of 2,962). The peptide corresponding to residues 73 to 81 (incorporating one Cys residue), expected mass of 1,148, was detected as a 2+ ion of mass/charge 574 in the control incubation and not in the drug-incubated sample. For the latter, a 2+ ion of mass/charge 696, which would correspond to the peptide fragment comprising residues 73 to 81 plus one bound molecule of 1 (expected calculated mass of 1,391) was detected (results not shown).
Insulin reduction. The ability of quinol analogues to inhibit TR/thioredoxin–mediated signal transduction was examined exploiting the ability of this pathway to reduce insulin. When reaction mixtures including all reagents were incubated for 30 minutes at 37°C, analogues 1 and 2 inhibited insulin reduction dose-dependently, yielding IC50 values of 28 and 37.14 µmol/L, respectively (Fig. 8). Extended incubation times (60 minutes) failed to have any notable effect on insulin reduction. Interestingly, when reaction mixtures, excluding insulin were incubated for 30 minutes (37°C), then a further 30 minutes upon addition of insulin, insulin reduction by TR/thioredoxin signal transduction in the presence of these molecules was significantly decreased (IC50 values 2.68 and 5.95 µmol/L, respectively, were obtained for 1 and 2; Ps < 0.001). These comparative observations concur exactly with previous experiments using Escherichia coli thioredoxin and TR as signal transducers for the reduction of insulin (results not shown).
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Discussion |
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,ß-unsaturated carbonyl function able to form covalent bonds with sulfur nucleophiles (thiols) at the ß-carbon position. Pearson correlation coefficients (>0.7), suggested mechanistic similarity between 1 and these disparate structures that elicited toxicity in colon, renal, and certain breast cell lines (6). Thus, a platform for interrogation of the database for molecular targets expressed in different cell types was provided. Of interest was TR/thioredoxin signal transduction. A working model proposed covalent binding between 1 and one thiol residue of Cys (32 or 35) of thioredoxin, to give a reversible monosulfur adduct, followed by addition of a second Cys thiol residue to form an irreversible complex (8). Structural studies comparing the Cys 32-Cys 35 sulfur interatomic distance in human thioredoxin with the interatomic distance between the electrophilic ß-carbon atoms of the quinol pharmacophore provided evidence to support the model (9). Indeed, MS generated irrefutable evidence of binding between of human thioredoxin and up to five molecules of 1 (Fig. 6). By performing trypsin digests, interactions between Cys residues of human thioredoxin and this small molecule were determined (Fig. 7). MS/MS verified the formation of covalent adducts between 1 and thioalkyl residues of thioredoxin, in the presence of the competing dithiol DTT. Such observations are consistent with our proposal that an irreversible reaction covalently binds 1 with reduced thioredoxin, the consequence of which would be inhibition of signal transduction. Indeed, powerful dose-dependent inhibition of TR/thioredoxin-catalyzed reduction of insulin (IC50 < 6 µmol/L) by 1 and 2, in a manner concordant with irreversible thioredoxin inhibition, has been shown (Fig. 8).
Thioredoxin, a 12-kDa protein, is a potent intracellular disulfide reductase possessing key roles in the regulation of biological functions such as cellular proliferation (deoxyribonucleotide biosynthesis), growth control, and apoptosis. Cytoplasmic/nuclear thioredoxin, discussed herein, together with mitochondrial thioredoxin-2 serve to protect the cell against oxidative stress and both are essential for embryonic development (10). Thioredoxin, an important mediator of redox regulation also modulates the actions of crucial cellular enzymes and transcription factors (11, 12). Thioredoxin interacts with redox factor-1 (Ref-1), modifying the binding activity of activator protein-1 (AP-1). Thioredoxin activates NF
B evoking cellular responses to oxidative stress, tumorigenesis, and apoptosis. Reduced thioredoxin prevents apoptosis by complexing apoptosis signal regulating kinase-1 (ASK-1). When thioredoxin is oxidized by reactive oxygen species, binding between ASK-1 and thioredoxin dissociates, and apoptosis signal transduction is activated (13). Elevated thioredoxin and TR, found in many human tumors including lung, colon, hepatoma, and pancreas (14, 15), are associated with poor prognosis and decreased patient survival. Thioredoxin may act as a growth factor and promote aggressive tumor growth, angiogenesis, inhibit apoptosis, and augment resistance within tumors to chemotherapeutic agents such as cisplatin. Thioredoxin regulates HIF-1 protein levels under normoxia and hypoxia. Thioredoxin transfection has been shown to elicit significantly elevated hypoxia-induced HIF-1 transactivation activity resulting in a significant increase in the protein products of hypoxia-responsive genes such as vascular endothelial growth factor (VEGF). Indeed, VEGF production by tumor cells was reduced by 1, and colorectal carcinoma cell lines showed enhanced sensitivity to 1 under hypoxic conditions (16).
Gene array analyses of HCT 116 colon carcinoma cells showed that of 10,000 cancer-related genes, expression of only one, TR, was enhanced >4-fold following exposure of cells to 1 (1 µmol/L) for 24 hours. Following treatment of HCT 116 cells with 2, the most significantly induced gene (>3-fold increase in expression) was again TR (Fig. 4). Such up-regulation of TR suggests an attempt by the cell to compensate for the enhanced oxidative stress inhibition of thioredoxin would trigger, and provides further corroboration of a mechanism of action that includes inhibition of thioredoxin. Indeed, transcription of HO-1, a protein highly induced in response to oxidative stress (17), was increased dose-dependently by 2.
Whereas our evidence supports molecular interaction between heteroaromatic quinols and thioredoxin, we acknowledge the probable involvement of other targets and mechanisms in antitumor activity. For example, protein-disulfide isomerases (PDI), which offer protection against apoptosis and provide chaperone activity, show sequence and structural homology to thioredoxin possessing two independent catalytic sites for thiol-disulfide bond exchange reactions (18). Endothelial cell-specific PDI (EndoPDI) has three thioredoxin active site motifs, whose Cys residues may be susceptible to covalent interaction with quinol analogues. Indeed, experiments investigating interactions between 1 and EndoPDI are under way.5 EndoPDI, present in tumor endothelium, but rarely expressed in normal tissues, is induced by hypoxia, acting as a stress survival factor (18). Growth inhibition of proliferating human vein endothelial cells by 1 has been shown (16), tube formation was aborted, suggesting inhibition of endothelial differentiation, an observation also consistent with the antiangiogenic activity of 1.
Microarrays showed down-regulation of cdk-1 gene expression, translating to dose-dependent decreases in CDK-1 protein expression (Fig. 5) in lysates of cells treated with 1 or 2, and accompanied by profound perturbation in cell cycle profiles (Fig. 3). CDK1 is integral to G2-M progression and its expression has been shown to be under the partial control of AP-1 elements within its promoter (19). When coupled with the ability of thioredoxin to activate AP-1 through direct interaction with DNA repair protein and Ref-1 (20), it could be argued that inhibition of thioredoxin activity may decrease AP-1 transcription of the cdk-1 gene. Transcription of GADD45, whose protein product accumulates following DNA damage and is implicated in cell cycle control and DNA repair in response to genotoxic stress, is up-regulated by 1. GADD45 causes G2-M cell cycle arrest through direct interaction and disruption of CDK-1/cyclin B1 complexes (21). 7-Hydroxystaurosporine (UCN-01), the DNA repair inhibitor, capable of abrogating G2 arrest (22–24) via a mechanism which includes inhibition of CDK-1 phosphorylation, failed to influence G2-M cell cycle blocks induced by 1 and 2. Similarly, cotreatment of cells with UCN-01 and 1 or 2 did not potentiate quinol cytotoxicity (results not shown). Finally, transcription of asparagine synthetase was down-regulated by 1 and 10 µmol/L 1, raising the possibility that L-asparagine amidohydrolase (asparaginase) and 1 may represent an effective experimental treatment combination.
We believe that modulation of thioredoxin represents a valid therapeutic goal; indeed, one other inhibitor of thioredoxin is currently undergoing clinical evaluation. The disulfide PX12, irreversibly thioalkylates the noncatalytic site Cys73 of thioredoxin, thereby disabling its ability to be reduced by TR (25). Thus, compounds 1 and 2 represent a distinct mechanistic class of thioredoxin inhibitor; they are synthetically accessible, and show selectivity and potency against breast, colon, and renal cell lines in vitro and in vivo. Importantly, we have shown that this class of molecule targets thioredoxin, a protein possessing key roles in the etiology of malignant disease.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank NCI for collaborations and for critical appraisal of the article, Stewart Martin, and Abhik Mukherjee.
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Footnotes |
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3 http://www.cancerresearchuk.org 
4 http://apps1.niaid.nih.gov/david/
5 A. Mukherjee, personal communication.
Received 11/18/04. Revised 2/ 3/05. Accepted 2/16/05.
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