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. 2008 Apr 21;154(5):940–948. doi: 10.1038/bjp.2008.161

Arsenic trioxide and auranofin inhibit selenoprotein synthesis: implications for chemotherapy for acute promyelocytic leukaemia

S Talbot 1, R Nelson 1, W T Self 1,*
PMCID: PMC2451042  PMID: 18587442

Abstract

Background and purpose:

Arsenicals have been used medicinally for decades to treat both infectious disease and cancer. Arsenic trioxide (As2O3) is effective for treatment of acute promyelocytic leukaemia (APL), yet the mechanism of action of this drug is still widely debated. Recently, As2O3 was shown to inhibit the activity of the selenoenzyme thioredoxin reductase (TrxR). TrxR has been proposed to be required for selenium metabolism. The effect of inhibitors of TrxR on selenium metabolism has yet to be assessed. This study aims to determine whether chemotherapeutics that target selenocysteine within selenoenzymes may also affect the metabolism of selenium.

Experimental approach:

A lung cell line, A549, was used to assess the effect of TrxR inhibitors on selenium metabolism, using 75Se-selenite. The level of mRNA encoding cytosolic TrxR (TrxR1) was determined using real-time reverse transcriptase-PCR. TrxR activity was determined in whole-cell extracts.

Key results:

Exposure of cells to As2O3, arsenite or auranofin led to a concentration-dependent reduction of selenium metabolism into selenoproteins. Knockdown of TrxR1, using small inhibitory RNA, did not affect selenium metabolism. Exposure of cells to monomethylarsonic acid, a potent inhibitor of TrxR, did not alter selenium metabolism but did inhibit enzyme activity.

Conclusions and implications:

As2O3 and auranofin block the metabolism of selenium in A549 cells. Because As2O3 is used to treat APL, our findings may reveal the mechanism of this therapeutic action and lead to further research targeting selenium metabolism to find novel chemotherapeutic agents for the treatment of APL.

Keywords: arsenic trioxide, arsenite, auranofin, thioredoxin reductase, selenium, selenite, selenoprotein, acute promyelocytic leukaemia

Introduction

Environmental exposure to arsenic has been associated with the development of cancer of the lung, bladder, liver, kidney and skin (Hughes, 2002). The molecular mechanisms by which arsenicals contribute to carcinogenesis have been elusive and many hypotheses are being tested (Hughes, 2002; Kitchin and Ahmad, 2003). Trivalent arsenicals have been shown to induce oxidative stress in cell culture and animal models, and this oxidative damage is now seen as a critical link between arsenic exposure and cancer (Kitchin and Ahmad, 2003). Hydrogen peroxide has been implicated as the primary oxidant that increases upon exposure to arsenite (Pi et al., 2003); however, there is also evidence that superoxide and hydroxyl radicals increase (Shi et al., 2004). The binding of trivalent arsenicals to vicinal thiols has long been a hallmark of these compounds and indeed is likely to be one of the possible mechanisms for this oxidative stress (Hughes, 2002). Even the classic inhibition of pyruvate dehydrogenase has now been proposed to be due to oxidative stress (Samikkannu et al., 2003).

Arsenic is most often studied for its contribution to carcinogenesis, although arsenicals have been used medicinally for centuries (Waxman and Anderson, 2001; Dilda and Hogg, 2007). The trivalent form of arsenic, arsenic trioxide (As2O3), remains a vital tool in the treatment of acute promyelocytic leukaemia (APL) (Miller et al., 2002; Woo et al., 2002). Although arsenicals have been used medicinally for treating both APL and infectious diseases, the molecular mechanism(s) of these metalloid-based drugs remains poorly understood (Waxman and Anderson, 2001). Several clinical trials are ongoing to test the efficacy of As2O3 against solid tumours either in conjunction with all trans-retinoic acid or alone. It is well established that As2O3 reacts with thiols in the cell to form glutathione conjugates (Rey et al., 2004) and subsequently will also interact with closely spaced protein thiols (Woo et al., 2002; Samikkannu et al., 2003). A recent report has demonstrated that As2O3 can inhibit the active site of the selenoenzyme thioredoxin reductase (TrxR) (Lu et al., 2007). Arsenite and the methylated trivalent arsenical monomethylarsonic acid (MMAIII) have also been shown to be potent inhibitors of TrxR both in vitro and in vivo (Lin et al., 1999, 2001; Chouchane and Snow, 2001).

Selenium, also a toxic metalloid, has been studied in recent years more for its need as a critical micronutrient in the mammalian diet (Stadtman, 2000). Selenium is required for the synthesis of 25 selenoproteins that contain selenocysteine (Gromer et al., 2005). The two best-studied mammalian selenoenzymes are TrxR and glutathione peroxidase. Several isoenzymes exist for both, and selenocysteine is required for activity in both enzymes. The primary role of cytosolic TrxR (TrxR1) is to reduce thioredoxin (Trx). Reduced Trx is the source of reducing potential for DNA synthesis, methionine sulphoxide reductases and peroxiredoxins (Arner and Holmgren, 2000). Glutathione peroxidase isoenzymes play a critical role in reducing both hydrogen peroxides and lipid peroxides throughout the cell (Flohe et al., 1973, 2000; Ursini et al., 1995). Because of its presence in these enzymes, selenium is a critical micronutrient both for defending against oxidative stress and in cellular proliferation.

As TrxR has such a key role in proliferation and in defence against oxidative stress, several recent studies have attempted to validate inhibition of TrxR as a novel cancer therapy (Urig and Becker, 2006). Both Trx and TrxR were found to be upregulated in tumour-derived cell lines, helping to validate TrxR as a potential target (Berggren et al., 1996). Both novel natural products and platinum compounds have been found to inhibit TrxR1 (Becker et al., 2001; Wipf et al., 2001). The combination of oxidative stress and a lack of DNA synthesis could indeed be a potent weapon for cancer chemotherapy, as indicated by more recent studies using a gadolinium compound that shows promise in clinical trials (Hashemy et al., 2006). Gold-based drugs have been established inhibitors of TrxR enzymes in vitro for several years (Hill et al., 1997; Gromer et al., 1998; Rigobello et al., 2002; Engman et al., 2006; Omata et al., 2006). The gold compound, auranofin, used to treat rheumatoid arthritis, has been shown to be the most potent inhibitor of this enzyme activity in vitro and in vivo (Rigobello et al., 2002; Omata et al., 2006). Both have a high affinity for the active site of selenol (reduced form of selenocysteine) in the penultimate CXU (cysteine, any amino acid, selenocysteine) motif (Gladyshev et al., 1996).

Selenocysteine is inserted into the polypeptide chain by a series of specific elongation factors and through the use of an RNA stem–loop structure known as a SECIS (selenocysteine insertion sequence) element (Berry et al., 2001; Carlson et al., 2004; Hoffmann and Berry, 2005; de Jesus et al., 2006). In both eukaryotes and prokaryotes, selenium must first be reduced to hydrogen selenide (Se2−) before activation by an enzyme termed selenophosphate synthetase (Ehrenreich et al., 1992; Low et al., 1995; Yuan et al., 2006). The mechanisms by which selenium is taken up into cells and subsequently reduced are poorly understood, yet a chemical reaction known as the Painter reaction has been shown to efficiently reduce selenite to selenide in the presence of excess thiols (Ganther, 1968, 1971, 1999). Because of the need for a strong reducing potential to maintain selenium in the form of selenide, it has been suggested that TrxR plays a role in selenium metabolism (Arner and Holmgren, 2000). Indeed, it has been shown that TrxR can efficiently reduce selenite to selenide in vitro (Bjornstedt et al., 1997). A putative role for TrxR in selenium metabolism has been postulated based on this study, but no evidence for this occurring in vivo has been reported.

In addition to affecting TrxR activity, trivalent arsenicals have also been shown to affect the metabolism of selenium. The initial report by Moxon (1938) demonstrated a reduction in toxicity from selenium poisoning by administration of arsenite. This mutual sparing effect was later named the Moxon effect and has been studied primarily in animal models over several decades (Levander and Baumann, 1966; Levander, 1977). A metabolic link between arsenic and selenium was established in a key finding by Gailer et al. (2000b, 2002). A stable compound, seleno-bis(S-glutathionyl) arsinium ion, is formed upon reaction of arsenite with reduced glutathione, followed by reaction with hydrogen selenide. Similarly, studies have shown that administration of selenium to animals can affect the metabolism and distribution of mercury (Cikrt and Bencko, 1989; Urano et al., 1997; Gailer et al., 2000a; Gregus et al., 2001), and evidence has been shown that the coexcretion of mercury and selenium is tied to the formation of thiol conjugates. Platinum and gold drugs have been shown to affect selenium metabolism in animal models as well (Gregus et al., 2000). Therefore, there is a growing body of evidence that many of the metals that function to inhibit the reduced selenol of TrxR may also react with other reduced forms of selenium. Given the surge of interest in targeting TrxR, essentially through its reduced selenol residue, one must both determine the role that this enzyme plays in selenium metabolism and also appreciate the potential chemistry that can occur between reactive metals and selenium metabolic intermediates.

In this study, we have determined the impact of two clinically relevant inhibitors of TrxR, As2O3 and auranofin, on selenium metabolism in a cultured lung cell line. We have also evaluated the role that TrxR plays in the reduction and metabolism of selenium in the same culture model.

Methods

Cell culture

A549 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modification of Eagle's medium (DMEM) supplemented with L-glutamine, sodium pyruvate, 4.5 g L−1 glucose, 100 μg mL−1 streptomycin, 100 IU mL−1 penicillin (Mediatech, Herndon, VA, USA) and 10% fetal bovine serum (Equi-tech Bio, Kerrville, TX, USA). Cells were maintained in a humidified incubator at a temperature of 37 °C under an atmosphere of 5% CO2.

Radioisotope labelling

To follow incorporation of selenium into selenoproteins, A549 cells were cultured in six-well plates and grown to approximately 70% confluence. Radioisotope 75Se (2 μCi), carried with 10 nM of unlabelled sodium selenite, was added to each culture. Exposure to arsenite (0, 2, 6 or 10 μM), As2O3 (0, 1, 2.5 or 5 μM) or MMAIII (0, 2 or 6 μM) immediately preceded the addition of the radioisotope. In studies following the effects of auranofin on selenium metabolism, the addition of auranofin (0.1, 0.25, 1 or 3 μM) to cells was performed 4 h before addition of either 75Se or 35S. The cells were incubated in the presence of radioisotope selenium for 24 h before harvesting. To monitor effects on protein synthesis, 30 μCi of 35S, in the form of a cysteine/methionine mixture, was added to each culture under the same conditions described above for the labelling of selenoproteins. In these experiments, cells were cultured in a cysteine/methionine-depleted DMEM (Mediatech).

Cells were harvested by first washing with Dulbecco's phosphate-buffered saline, and then the monolayer was disrupted by the addition of trypsin-EDTA (Mediatech) for 4 min at 37 °C. Cells were collected by centrifugation and subsequently washed with Dulbecco's phosphate-buffered saline. The resulting cell pellets were resuspended in 200 μL of lysis buffer (50 mM tricine, pH 8.0, 0.1 mM benzamidine, 0.5 mM EDTA and 1 mM dithiothreitol) and lysed by sonication for 6–8 s using a model 100 sonic dismembrator (Thermo Fisher Scientific, Pittsburgh, PA, USA) at a power setting of 4 W. Crude lysates were clarified by centrifugation at 15 700 g for 7 min at 4 °C. The amount of 75Se was determined in extracts using a Wallac Wizard Gamma Counter, model 1470 (Perkin Elmer, Waltham, MA, USA) and 35S by liquid scintillation with a Packard TriCarb 2900TR counter (Perkin Elmer). Protein concentration was quantified by the method of Bradford (1976) using BSA as a standard. To confirm that selenium incorporation into specific selenoproteins occurred, 25 μg of protein was separated using a 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, dried and then exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, CA, USA). Selenoproteins were visualized using a Storm Phosphoimager and analysed using ImageQuant software (Molecular Dynamics).

Transient small inhibitory RNA knockdowns

A549 cells were seeded in six-well plates and grown to approximately 60% confluence. Small inhibitory RNA (siRNA) duplex molecules specifically targeting the mRNA encoding p42 microtubule-associated protein kinase (Erk2 (extracellular signal-regulated kinase)), Trx and TrxR1 were obtained from Qiagen (Valencia, CA, USA). The sequences (sense and antisense RNA) used are as follows (5′ to 3′): Erk2-UGCUGACUCCAAAGCUCUG, CAGAGCUUUGGAGUCAGCA; Trx-CCAUUAAUGAAUUAGUCUA, UAGACUAAUUCAUUAAUGG; TrxR1-GCAAGACUCUCGAAAUUAU, AUAAUUUCGAGAGUCUUGC. A non-silencing fluorescent control oligonucleotide duplex, labelled with Alexa Fluor 488, was used to determine siRNA transfection efficiency. The efficiency of transfection with this fluorescent siRNA was determined to be 66% (s.d.: ±9%) by directly counting fluorescent cells using an inverted fluorescence microscope (Zeiss, Thornwood, NY, USA).

Transfection complexes were prepared by a mixture of serum-free media, HiPerfect transfection reagent (Qiagen) and 5 nM siRNA. These reagents were incubated for 10 min at room temperature to allow transfection complexes to form and were subsequently added dropwise to cells for optimal transfection efficiency. Immediately following the addition of 5 nM siRNA, cells were treated with 3 μM auranofin to inhibit existing TrxR. Cells were incubated for 24 h with siRNA and auranofin before removing media and labelling with 75Se, as described in the previous section.

Real-time reverse transcriptase-PCR analysis

For semiquantitative analysis of mRNA, cells were cultured in 25 cm2 flasks and treated with arsenite, As2O3 or auranofin for 24 h at the appropriate concentrations. Cells were harvested by washing with Dulbecco's phosphate-buffered saline and subsequently removed from the monolayer using trypsin-EDTA. The cells were then washed with Dulbecco's phosphate-buffered saline treated with 0.1% diethylpyrocarbonate. RNA was isolated using a ChargeSwitch Total RNA kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions, and the RNA concentration was determined by UV-visible spectrophotometry at 260 nm using an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). cDNA was produced from 0.5 μg of RNA as a template using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions.

Primers used for real-time reverse transcriptase-PCR (RT-PCR) included (listed forward then reverse) TrxR1: 5′-AGCTCAGTCCACCAATAGTGA-3′ and 5′-GGTATTTTTCCAGTCTTTTCAT-3′; β-actin: 5′-CATGTACGTTGCTATCCA-3′ and 5′-CTCCTTAATGTCACGCACGAT-3′; Trx: 5′-GCAGATCGAGAGCAAGACTG-3′ and 5′-CTCCAGAAAATTCACCCACC-3′. All real-time RT-PCR was carried out in a Bio-Rad I-Cycler (Bio-Rad), and reactions (total volume of 25 μL) consisted of SYBRgreen supermix (Bio-Rad), four oligonucleotides at a final concentration of 0.2 mM each, and forward and reverse primers at a final concentration of 0.2 μM and 5 μL of 1:100 diluted cDNA. Reaction conditions were as follows: one cycle at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 55 °C for 45 s. Melt curves were used to determine the formation of a single product. Efficiency of amplification of each primer pair was calculated with a 10-fold dilution series of control cDNA. The relative expression (fold change in expression versus control) for each of the mRNAs of interest using β-actin as an internal mRNA standard was determined using a previously described method (Pfaffl, 2001).

TrxR activity assays

A549 cells were cultured in 75 cm2 flasks in DMEM with 10% serum as described above. Cells were treated with arsenite, MMAIII or auranofin for 48 h before to harvesting with the appropriate concentrations of each compound. Cell pellets were resuspended in lysis buffer (5 mM potassium phosphate (pH 7.4) and 0.5 mM EDTA) and sonicated using a model 100 sonic dismembranator (Thermo Fisher Scientific) with a power of 4 W. TrxR assays were performed as described previously with minor modifications (Smith and Levander, 2002). To measure the gold-inhibited activity to differentiate TrxR from glutathione reductase, cell extracts were preincubated with 1 μM auranofin for 20 min at room temperature to allow for complete inhibition of TrxR. A 50 μg portion of protein was used to initiate the activity assay in reactions in a 96-well plate. Reduction of 5,5′-dithiobis-(2-nitrobenzoic acid) was followed at 412 nm using a SpectraMax 190 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Reactions were carried out at 37 °C, taking readings every 15 s for 3 min. TrxR activity is reported as previously described (Smith and Levander, 2002).

Statistical analysis

All statistical analyses were performed using Microsoft Excel, and ANOVA was performed to test for statistical significance within data sets.

Materials

Sodium selenite, sodium arsenite and As2O3 were obtained from Acros Organics (Geel, Belgium). MMAIII was from the laboratory of Dr. William Cullen (University of British Columbia, Vancouver, BC, Canada). The 75Se radioisotope was obtained from the University of Missouri Research Reactor (MURR, Columbia, MO, USA) in the form of selenious acid. 35S-methionine/cysteine label was obtained either from Amersham BioSciences (Piscataway, NJ, USA) or from Perkin Elmer Life Sciences (Boston, MA, USA). Auranofin was from Axxora LLC (San Diego, CA, USA).

Results

Arsenite inhibits selenoprotein synthesis in lung adenocarcinoma cells (A549)

Addition of arsenite at concentrations between 2 and 10 μM resulted in a concentration-dependent decrease in selenoprotein synthesis in A549 cells. We separately tested for the cytotoxicity of arsenite after incubation with A549 for 24 h using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (data not shown). A 50% reduction in cell viability was observed when cells were incubated with 100 μM arsenite. At concentrations of arsenite at or below 10 μM, no significant changes in cell viability were observed. Exposure of cells to arsenite did slightly reduce protein synthesis in cells treated at all three concentrations (Figure 1b); however, there was no dose-dependent decrease as is seen for selenoprotein synthesis.

Figure 1.

Figure 1

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Treatment of lung adenocarcinoma cells (A549) with arsenite inhibits selenoprotein synthesis. A549 cells were cultured in DMEM and then exposed to arsenite (0, 2, 6 and 10 μM), immediately followed by radiolabelling with 75Se (selenite) (a) or 35S (b) for 24 h. Cells were subsequently harvested and 30 μg of protein from cell extracts was separated by 15% SDS-PAGE. Proteins were visualized by autoradiography. TrxR was identified as the predominant labelled selenoprotein by its size using a standard protein marker (data not shown). Cells were treated for 24 h with 0, 2 or 10 μM arsenite followed by isolation of RNA for real-time RT-PCR analysis. β-Actin was used as an internal standard. Mean relative expression (fold) is plotted from multiple experiments with cultures grown in triplicate in each experiment. Error bars represent the s.d. No statistical significance was found between TrxR1 transcript levels in cells treated with arsenite, as determined by one-way ANOVA (P>0.05) (c). DMEM, Dulbecco's modification of Eagle's medium; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; TrxR, thioredoxin reductase; RT, reverse transcriptase.

As overall selenoprotein synthesis is reduced in cells treated with arsenite, we determined whether this treatment was significantly affecting the stability of mRNAs encoding selenoproteins. TrxR1 is the most abundant selenoprotein expressed in A549 (Figure 1a), hence we chose its mRNA for this analysis. Using semiquantitative real-time RT-PCR, we evaluated the impact of treating cells with arsenite on TrxR1 mRNA levels. Figure 1c shows that no statistically significant changes in TrxR1 mRNA levels were observed, yet a slight increase was seen in cells treated with 10 μM arsenite.

These results, when combined with radiolabelling data in Figure 1a, clearly show that subtoxic levels of arsenite significantly reduce the incorporation of selenium into selenoproteins in lung adenocarcinoma cells. Two mechanisms could account for this effect: the direct formation of seleno-bis(S-glutathionyl) arsinium ion, as described by Gailer et al. (2000b), or the inhibition of TrxR, as implicated in selenoprotein synthesis, as described by Holmgren (Bjornstedt et al., 1997; Arner and Holmgren, 2000). As the impact of trivalent inorganic arsenicals on human cells has implications both in carcinogenesis and chemotherapy, we designed experiments to determine which of the above mechanisms are most likely to account for the observed inhibition of selenium metabolism.

As2O3 blocks selenium incorporation into selenoproteins

Similar to the effects of arsenite, As2O3 treatment of A549 cells led to decreases in selenoprotein synthesis as demonstrated by 75Se radiolabelling (Figure 2a). This inhibition was observed in cells treated with As2O3 at a concentration of 2.5 μM and continues in a concentration-dependent manner. In addition, As2O3 did not affect general protein synthesis, as determined by 35S labelling studies (Figure 2b). Likewise, as with treatment with arsenite, mRNA levels encoding TrxR1 do not significantly change with increasing exposure to As2O3 (Figure 2c). Given that two trivalent arsenicals, known inhibitors of TrxR1, decrease selenium incorporation into selenoproteins, the next step was to see if this phenotype was again present with a separate inhibitor of TrxR1 that is not arsenic based. For this, we chose the gold-containing compound, auranofin.

Figure 2.

Figure 2

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As2O3 (ATO) inhibits selenoprotein synthesis but not general protein synthesis in A549 cells. A549 cells were cultured in DMEM and treated with either 0, 1, 2.5 or 5 μM ATO and labelled with 2 μCi of 75Se (a) or 35S (b) for 48 h. Cells were harvested and 15 μg of protein from extracts was separated by SDS-PAGE (15%). Selenoproteins (75Se) and general protein synthesis (35S) were visualized by phosphorimage analysis. For RT-PCR, (c) cells were exposed to 0, 1 or 5 μM ATO for 48 h. Mean relative expression (fold) is plotted from multiple experiments with cultures grown in triplicate in each experiment. Error bars represent the s.d. No statistical significance was found between TrxR1 transcript levels in cells treated with arsenite, as determined by one-way ANOVA (P>0.05) (c). DMEM, Dulbecco's modification of Eagle's medium; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; RT, reverse transcriptase.

Auranofin treatment of A549 cells inhibits selenoprotein synthesis

Auranofin has been shown to inhibit TrxR both in vitro and in cultured cells (Gromer et al., 1998, 2002; Rigobello et al., 2002, 2005; Omata et al., 2006). Treatment of A549 cells with auranofin at concentrations ranging from 0.1 to 3 μM resulted in dramatic decreases in selenoprotein synthesis (Figure 3a). Treatment with as little as 1 μM auranofin led to a near complete cessation of selenium incorporation into newly synthesized selenoproteins. To confirm that this effect was not due to the inhibition of protein synthesis in general, we followed incorporation of 35S into proteins under the same experimental conditions (Figure 3b). No significant changes in protein synthesis occurred, demonstrating that the impact of auranofin was specific to selenium metabolism. The treatment of cells with auranofin also did not affect mRNA levels encoding for TrxR1 (Figure 3c). These results further support the hypothesis that the mode of action of arsenite and As2O3 is through a direct inhibition of TrxR.

Figure 3.

Figure 3

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Auranofin treatment results in inhibition of incorporation of selenium into selenoproteins. A549 cells were treated with varying concentrations of auranofin (0, 0.1, 0.25, 1 and 3 μM) and protein synthesis was followed with either 75Se (a) or 35S (b) for 24 h. Cell extracts were separated by 15% SDS-PAGE and visualized by autoradiography. For RT-PCR, (c) cells were exposed to 0, 0.25 or 3 μM auranofin for 24 h. Mean relative expression (fold) was plotted from multiple experiments from cultures grown in triplicate in each experiment. Error bars represent the s.d. No statistical significance was found between the TrxR1 transcript levels in cells treated with arsenite as determined by one-way ANOVA (P>0.05) (c). SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; RT, reverse transcriptase.

Auranofin and other gold compounds have been previously shown to inhibit TrxR1 in cultured cells (Omata et al., 2006). We wanted to confirm that inhibition of TrxR1 was indeed occurring under our treatment conditions in A549. Auranofin treatment of A549 cells did result in inhibition of TrxR activity (Table 1); however, the residual TrxR activity present in cells treated with 1 and 3 μM auranofin did not directly correlate with the potency observed in the selenium labelling experiment (Figure 3a). When TrxR activity was reduced only slightly, specifically in cells treated with 1 μM auranofin (Table 1), the incorporation of selenium into selenoproteins was dramatically reduced (Figure 3a). This finding suggests that TrxR1 may not be the primary target of arsenite and also suggests that auranofin may inhibit selenium metabolism through an alternative, as yet undescribed, mechanism.

Table 1.

TrxR activity in cells treated with auranofin

Concentration of auranofin (μM) TrxR activity (nmol min−1 mg−1) s.d.
0 10.4 1.86
0.25 10.3 1.83
1 7.82 1.19
3 1.99 0.81
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Abbreviations: DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); TrxR, thioredoxin reductase.

TrxR activities were determined by DTNB assay, as previously described. A549 cells were cultured for 24 h in the presence of 0, 0.25, 1 or 3 μM auranofin before harvesting. The average TrxR activity is derived from six independent experiments with duplicate cultures, and the s.d. from the mean is shown.

Knockdown of Trx or TrxR1 does not result in changes in selenoprotein synthesis

To directly assess the role of TrxR1 on selenoprotein synthesis, we utilized transient transfection of siRNA molecules that specifically target TrxR1, Trx or a negative control (microtubule-associated protein kinase-1/Erk2) to critically evaluate the role of the TrxR/Trx system on selenium metabolism. Because of the need for potent reducing potential for the efficient conversion of selenite to selenide, it had been previously suggested that this system was required for selenium metabolism to selenide (Bjornstedt et al., 1997; Arner and Holmgren, 2000). There is good biochemical evidence that TrxR can efficiently reduce selenite to selenide in vitro, but there is no evidence for the reduction of selenite to selenide in cell culture or animal studies.

As expected, knockdown of microtubule-associated protein kinase-1 did not affect selenoprotein synthesis (Figure 4, lane 2). Knockdown of the mRNA encoding Trx was confirmed by real-time RT-PCR (Figure 5a), but it did not affect the incorporation of selenium into other selenoproteins (Figure 4a, lane 3). Surprisingly, knockdown of TrxR1 did not affect overall selenoprotein synthesis, although no new synthesis of TrxR1 was taking place based on selenium 75Se incorporation (Figure 4, lane 4). For the experiment shown in Figure 4, cells were pretreated with auranofin at 3 μM to efficiently reduce TrxR1 activity (residual) before and during the early phases of the RNA interference transfection. This was performed in an attempt to remove existing TrxR1, so that a critical assessment of the role of TrxR1 could be accomplished. Omitting the pretreatment with auranofin did not alter the results obtained (data not shown). Knockdown of Trx and TrxR1 was confirmed by RT-PCR (Figure 5). These results strongly suggest that Trx and TrxR1 are not directly required for the reduction of selenium under these conditions.

Figure 4.

Figure 4

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Knockdown of Trx or TrxR1 does not affect incorporation of selenium into other selenoproteins. A549 cells were treated with 5 nM siRNA targeting the mRNA encoding microtubule-associated protein kinase-1 (MK), Trx or TrxR1. Cells were also treated with 3 μM auranofin to inhibit the activity of existing TrxR. After 24 h, the media were removed, replaced with fresh media without auranofin and 75Se was then added. The cells were then incubated for an additional 24 h to assess selenium incorporation. A 20 μg portion of protein from cell lysates was separated by 15% SDS-PAGE, and radiolabelled selenoproteins were visualized by 75Se autoradiography. Trx, thioredoxin; TrxR1, thioredoxin reductase 1; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Figure 5.

Figure 5

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Confirmation of Trx and TrxR1 knockdowns by RT-PCR. Real-time RT-PCR (a and b) was used to monitor efficiency of the Trx and TrxR1 knockdowns by analysing mRNA levels. β-Actin was used as an internal standard for analysis. Mean relative expression (fold change) in (a and b) is derived from a representative experiment with duplicate cultures that were analysed in triplicate. Error bars represent the s.d. Trx, thioredoxin; TrxR1, thioredoxin reductase 1; RT, reverse transcriptase.

MMAIII treatment of cells does not inhibit selenium incorporation into selenoproteins

Monomethylarsonic acid is known to be a potent inhibitor of TrxR and is probably the most potent inhibitor of TrxR1 of all trivalent arsenicals (Lin et al., 1999). If TrxR is involved in selenoprotein synthesis, then exposure of cells to MMAIII should generate the same phenotype as arsenite, As2O3 and auranofin. However, in cells treated with increasing concentrations of MMAIII and subsequently labelled with 75Se, there was no change in selenium incorporation into selenoproteins (Figure 6). Cells were exposed to a high enough concentration to inhibit the enzyme as determined by the 5,5′-dithiobis-(2-nitrobenzoic acid) assays (Table 2). Upon exposure to 6 μM MMAIII, TrxR is significantly inhibited and the level of selenium incorporation into selenoproteins was unchanged (Table 2 and Figure 5, lane 3). These results clearly show that inhibition of TrxR did not lead to inhibition of selenium metabolism.

Figure 6.

Figure 6

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MMAIII does not affect selenium incorporation into selenoproteins. Cells were exposed to 0, 2 or 6 μM MMAIII and immediately labelled with 75Se to follow selenium incorporation into selenoproteins for 24 h before harvesting. Approximately 20 μg of protein was separated by 15% SDS-PAGE. Selenoproteins were visualized by autoradiography. MMAIII, monomethylarsonic acid; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Table 2.

TrxR activity in A549 cells exposed to MMAIII

Concentration of MMAIII (μM) TrxR activity (nmol min−1 mg−1) s.d.
0 8.05 0.290
2 6.41 3.46
6 0.103 0.226
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Abbreviations: DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); MMAIII, monomethylarsonic acid; TrxR, thioredoxin reductase.

TrxR activities were determined by the DTNB assay, as described in the Methods section. Cells were cultured (six independent cultures) in duplicate with 0, 2 or 6 μM MMAIII for 48 h before harvesting.

Discussion and conclusion

Arsenic trioxide is currently approved for the treatment of APL; however, the mechanism of its carcinostatic action is not well understood. In a previous study, 2.5 and 5 μM As2O3 were found to inhibit TrxR1 in MCF-7 breast cancer cells by 40% (Lu et al., 2007). Here, we demonstrate that treatment of cells with As2O3 decreased selenium incorporation into selenoproteins. Our results show that arsenite or auranofin can also inhibit the incorporation of selenium into selenoproteins in lung cells, possibly through forming a complex as described by Gailer et al. (2002). Indeed, a recent study has shown that incubation of red blood cells with arsenite and selenite in vitro results in intracellular accumulation of this complex (Manley et al., 2006). The sole chemical requirement for synthesis of this complex is reduced glutathione, present in millimolar concentrations in nearly all mammalian cells. Our results may have implications for the mode of action of As2O3 in chemotherapy. With the decreased capacity to produce selenoproteins, the cell loses some of its antioxidant capabilities, and this could lead to increased oxidative stress. Oxidative stress has been suggested to be part of the mechanism of how As2O3 works in the treatment of APL. Our results would indicate that a metabolic inhibition of selenium metabolism could lead to this observed oxidative stress.

The mechanism of auranofin in the treatment of rheumatoid arthritis is also poorly understood. It has been reported to function by inhibiting nuclear factor-κB translocation to the nucleus (Yamashita et al., 2003). Several cell culture studies have also suggested that auranofin could be used for chemotherapy of tumours. In one such study, it was found that auranofin induced apoptosis in cisplatin-resistant ovarian cancer cells, mediated through the release of cytochrome c (Marzano et al., 2007). Auranofin was also shown to induce apoptosis in APL-derived cells when combined with retinoic acid (Kim et al., 2004). As our results show that auranofin reduces selenium incorporation into selenoproteins when administered to cells at a subtoxic level, this may suggest that both As2O3 and auranofin act to generate oxidative stress and arrest DNA synthesis through inhibition of selenium metabolism. This would then indicate that these results represent a ‘proof of principle' that blocking selenium metabolism could be a valid target for the development of novel chemotherapeutic agents.

There has been some speculation on the role of TrxR in selenoprotein synthesis (Ganther, 1999; Papp et al., 2006). Hydrogen selenide is required as the substrate for selenophosphate synthetase 2 to produce selenophosphate (Xu et al., 2006). We have recently shown that cells can transport selenide with high affinity (Ganyc and Self, 2008). This suggests that selenide metabolism is the critical link to selenium metabolism from uptake to initial activation by phosphorylation. In this study, we have demonstrated that TrxR is not involved in selenoprotein synthesis. Even though three known inhibitors of TrxR decreased selenium incorporation into selenoproteins, knockdown of TrxR1 expression resulted in no change in incorporation of selenium into selenoproteins. The targeted siRNA experiments suggest that TrxR1 is not involved in selenoprotein synthesis. However, exposure of cells to MMAIII, which blocked all TrxR activity, resulted in no change in incorporation of selenium into selenoproteins. It then becomes important to understand the possible molecular mechanisms of the inhibition of selenium metabolism induced by auranofin and As2O3.

A well-established metabolic inhibitor of selenium metabolism is arsenite (Gailer et al., 2000b, 2002). This metabolic inhibition occurs via the formation of seleno-bis(S-glutathionyl) arsinium ion (Gailer et al., 2002). We propose that inhibition of selenium metabolism is also occurring when cells are treated with As2O3 and auranofin, but not with MMAIII. The chemical nature of complexes formed between As2O3 or auranofin and selenide has not as yet been determined. Insoluble arsenic selenides have been studied for years as semiconductors as glasses (Chen et al., 2006); however, it is unclear as to whether an insoluble complex of arsenic and selenium is formed in vivo. Likewise, it is not yet known whether auranofin and selenide will form a stable conjugate. Studies are ongoing to define the metal/metalloid complexes formed between As2O3 or auranofin and selenide, and the results of this analysis should lead to an understanding of the inhibition of selenoprotein synthesis observed in this study. Indeed, we believe that the results shown in this report may lead to novel drug discovery efforts to target selenium metabolism as a means to treat APL.

Acknowledgments

We thank Drs Li Mei Chen and Karl Chai (Department of Molecular Biology and Microbiology, University of Central Florida) for their assistance with the fluorescent microscopy experiments. We also thank Dr William Cullen (University of British Columbia) for supplying MMAIII. This research was supported by grants to WTS from the National Institutes of Health (NIEHS ES01434) and the Florida Department of Health (05-NIR-10).

Abbreviations

APL

acute promyelocytic leukaemia

MMAIII

monomethylarsonic acid

TrxR

thioredoxin reductase

Conflict of interest

The authors state no conflict of interest.

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