Effects of the antioxidant Pycnogenol Ò on cellular redox systems in U1285 human lung carcinoma cells Valentina Gandin 1, *, Christina Nystro ¨ m 1 , Anna-Klara Rundlo ¨ f 1 , Kerstin Jo ¨ nsson-Videsa ¨ ter 2 , Frank Scho ¨ nlau 3 , Jarmo Ho ¨ rkko ¨ 4 , Mikael Bjo ¨ rnstedt 1 and Aristi P. Fernandes 1 1 Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden 2 Division of Haematology and Oncology, Department of Medicine, Karolinska University Hospital Huddinge, Stockholm, Sweden 3 Department of Pharmaceutical Chemistry, University of Mu ¨ nster, Germany 4 Almado, Turku, Finland Oxidative stress is associated with many pathological events, such as degenerative diseases, arteriosclerosis, inflammatory diseases and cancer. It is believed to be due to an imbalance in the formation and degradation of reactive oxygen species (ROS), e.g. superoxide anion (O 2 •) ), hydroxyl radical (OH •) ) and hydrogen peroxide (H 2 O 2 ) [1]. ROS are highly reactive compounds derived from oxygen, that in moderate amounts are needed for intracellular signalling, redox regulation and as a defence against infections. However, as the intracellular environment is normally reduced, an excessive amount of ROS can react with and harm important parts of the cell such as DNA, proteins and lipids. Pycnogenol Ò , a natural extract marketed as a food supplement or herbal drug, has been shown to have antioxidant and free radical scavenging activities [2]. Pycnogenol Ò is extracted from the bark of French mari- time pine (Pinus maritima), which grows in the coastal region of southwest France. The main constituents are monomeric phenolic compounds (catechin, epicatechin and taxifolin) and condensed flavonoids (procyanidines and proanthocyanidines). These compounds are also Keywords antioxidant; glutathione peroxidise; oxidative stress; Pycnogenol Ò ; thioredoxin reductase Correspondence A. P. Fernandes, Division of Pathology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, SE-14186 Stockholm, Sweden Fax: +46 8 58581020 Tel: +46 8 58582926 E-mail: aristi.fernandes@ki.se *Permanent address Department of Pharmaceutical Sciences, University of Padova, Italy (Received 29 August 2008, revised 15 October 2008, accepted 13 November 2008) doi:10.1111/j.1742-4658.2008.06800.x Pycnogenol Ò , which is extracted from the bark of French maritime pine, has been shown to have antioxidant and free radical scavenging activities. Thioredoxin reductase (TrxR), glutathione peroxidase (GPx) and glutathi- one reductase (GR) are three central redox enzymes that are active in endogenous defence against oxidative stress in the cell. Treatment of cells with Pycnogenol Ò decreased the activity of both TrxR and GPx in cells by more than 50%, but GR was not affected. As previously reported, both enzymes were induced after treatment with hydrogen peroxide and selenite. The presence of Pycnogenol Ò efficiently decreased selenite-mediated reac- tive oxygen species (ROS) production. Addition of Pycnogenol Ò after sele- nite treatment reduced the mRNA expression and activity of TrxR to basal levels. In contrast, the GPx activity was completely unaffected. The dis- crepancy between TrxR and GPx regulation may indicate that transcription of TrxR is induced primarily by oxidative stress. As TrxR is induced in various pathological conditions, including tumours and inflammatory condi- tions, decreased activity mediated by a non-toxic agent such as Pycnogenol Ò may be of great value. Abbreviations DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); GPx, glutathione peroxidase; GR, glutathione reductase; ROS, reactive oxygen species; SeMC, Se-methyl-seleno- L-cysteine; tert-BuOOH, t-butyl hydroperoxide; Trx, thioredoxin; TrxR, thioredoxin reductase. 532 FEBS Journal 276 (2009) 532–540 ª 2008 The Authors Journal compilation ª 2008 FEBS important constituents of fruits, vegetables and other plants [3]. Pycnogenol Ò has been shown to have excel- lent radical scavenger and antioxidant properties in model reactions that are superior to those of other fruit and plant extracts and other antioxidants [2,3]. Pycno- genol Ò has no mutagen activity according to the Ames test, and has low acute and chronic toxicity [2]. In addi- tion, Pycnogenol Ò has been shown to protect DNA against oxidative damage in vivo [4]. Supplementation with Pycnogenol Ò has been shown to have beneficial effects on patients with retinopathy, reducing retinal microbleeding and oedema and improving vision [5]. Pycnogenol Ò supplementation has also been shown to improve endothelial function in hypertensive patients, decreasing the hypertension [6], and to lower glucose levels in type 2 diabetic patients [7]. Thioredoxin reductase (TrxR), glutathione peroxi- dase (GPx) and glutathione reductase (GR) are three important redox enzymes in the endogenous defence against oxidative stress. As ROS are continuously formed in all aerobic organisms during metabolism, there is a need for effective defence systems that scav- enge the excessive amounts of ROS. Apart from being important in the defence against ROS, TrxR and GPx are selenium-containing enzymes [8]. Because it is part of important redox enzymes such as GPx and TrxR, selenium is an essential trace element, and considered a physiological antioxidant [9]. At low to moderate con- centrations, selenium is known to induce the expression of several selenoenzymes, including TrxR and GPx [10]. The biological effects of selenium are however strictly concentration-dependent, with antioxidant properties at low concentrations and powerful pro-oxidant effects at moderate to high concentrations. TrxR, together with thioredoxin (Trx) and NADPH, comprises an important defence system against oxida- tive stress, named the thioredoxin system [11,12]. Trx is a small ubiquitous protein with a redox-active disul- fide ⁄ dithiol that reduces disulfides, which is important in many redox-regulated reactions. TrxR is an essential protein that not only reduces Trx but also many other substrates such as selenium compounds [12] and Q10 [13]. Mammalian thioredoxin reductases are oxido- reductase flavoproteins, with a C-terminal active site with a conserved GCUG sequence [14,15] that is neces- sary for catalytic activity [16,17]. Three mammalian genes for TrxR have previously been identified. The first is the ‘classical’ cytosolic TrxR1 [18], with five potential isoforms that differ at the C-terminal. The other two, a mitochondrial and a glutaredoxin- containing thioredoxin reductase, are named TrxR2 and TGR, respectively [19–22]. The thioredoxin system is involved in many biological processes, such as DNA synthesis, apoptosis and regulation of several transcrip- tion factors [23]. Furthermore, it is known that thiore- doxin family proteins are induced in several tumors, and the thioredoxin system is thus suggested to be a prime target in cancer therapy [24–26]. The aim of this study was to investigate the antioxi- dant effects of Pycnogenol Ò on redox enzymes, with emphasis on the regulation of TrxR. Results Effects of Pycnogenol Ò on cellular TrxR enzyme activity To investigate the effects of Pycnogenol Ò on the TrxR, GPx and GR activity, the cells were cultivated for 48 h supplemented with various concentrations of Pycno- genol up to 25 lgÆmL )1 (Fig. 1A–C). The applied doses of Pycnogenol Ò (1–25 lgÆmL )1 ) did not signifi- cantly affect cell viability (Table 1). Cells were har- vested and homogenized and the enzyme activity was determined. Pycnogenol Ò treatment decreased the activity of both TrxR and GPx in cells by more than 50% compared to control cells, but the effect was less pronounced for GPx compared to TrxR (Fig. 1). In contrary to the selenoenzymes TrxR and GPx, Pycno- genol Ò did not affect the enzyme activity of GR (Fig. 1C). The reduction of TrxR and GPx activity was dose-dependent, with maximal effects achieved at concentrations of Pycnogenol Ò of 15 lgÆmL )1 for TrxR and 25 lgÆmL )1 for GPx (Fig. 1). Time-dependent effects of Pycnogenol Ò The time-dependent effect of Pycnogenol Ò was inves- tigated by cultivating U1285 cells with 25 lgÆmL )1 Pyc- nogenol Ò for 5 days. Cells were harvested at various time points, and the TrxR and GPx enzyme activity was measured (Fig. 2A,B). No time-dependent effects were observed, as the activity of both enzymes decreased in parallel with the duration of incubation for both treated and non-treated cells, reflecting varying expression at the various stages of the cell cycle. The presence of Pyc- nogenol Ò (25 lgÆmL )1 ) resulted in a remarked decrease in activity at all time points (Fig. 2). Dose-dependent effects of Pycnogenol Ò on TrxR enzyme activity in vitro To determine any direct inhibition of Pycnogenol Ò on TrxR enzyme activity, purified TrxR1 was incubated with Pycnogenol Ò at various concentrations (up to 100 lgÆmL )1 ) and for various durations (0, 5, 10, 15 V. Gandin et al. Effects of Pycnogenol Ò on redox systems FEBS Journal 276 (2009) 532–540 ª 2008 The Authors Journal compilation ª 2008 FEBS 533 and 30 min), and analysed spectrophotometrically. There was, however, no significant effect of Pycnoge- nol Ò on pure TrxR1 enzyme activity (data not shown). Effects on the viability of U1285 cells The toxicity of the compounds used was assessed by means of determination of the IC 50 value for each compound using a viability assay (Table 1). As shown in Table 1, the IC 50 of Pycnogenol Ò was first reached at doses exceeding 500 lgÆmL )1 . This clearly demon- A B C Fig. 1. Levels of activity after treatment with Pycnogenol Ò in U1285 cells. Activity of (A) TrxR, (B) GPx and (C) GR in U1285 cell homogenates treated for 48 h with increasing concentrations of Pycnogenol Ò (0–25 lgÆmL )1 ). Error bars represent the standard error based on at least three independent experiments. A B Fig. 2. TrxR and GPx activity in U1285 cell homogenates after treatment with Pycnogenol Ò for various time periods. (A) TrxR activity; (B) GPx activity. White bars indicate controls treated with 25 m M Hepes in NaCl ⁄ P i . Black bars indicate cells treated with 25 lgÆmL )1 Pycnogenol Ò . Error bars represent the standard error based on at least three independent experiments. Table 1. IC 50 values of various compounds in U1285 cells. Compound IC 50 (mean ± SD) a Pycnogenol Ò 511.12 ± 6.25 lgÆmL )1 Na 2 SeO 3 13.25 ± 3.12 lM Se-methyl-seleno-L-cysteine > 500 lM tert-butyl hydroperoxide 341.58 ± 9.75 lM a IC 50 values were calculated by probit analysis (P < 0.05; v 2 test). Effects of Pycnogenol Ò on redox systems V. Gandin et al. 534 FEBS Journal 276 (2009) 532–540 ª 2008 The Authors Journal compilation ª 2008 FEBS strates that the antioxidant properties of Pycnogenol Ò (1–10 lgÆmL )1 ) occur at concentrations far lower than the toxic dose. ROS production To further explore the antioxidant effect of Pycnoge- nol Ò , production of peroxide was measured in cells after treatment with 10 lgÆmL )1 Pycnogenol Ò in combination with either selenite, Se-methyl-seleno-l-cysteine (SeMC) or tert-butyl hydroperoxide (tert -BuOOH) (Fig. 3) at doses marginally affecting cell viability. These com- pounds are all known to cause oxidative stress, which is also shown in Fig. 3. Pycnogenol Ò was able to efficiently decrease ROS production by all compounds tested. Effect of Pycnogenol Ò on cellular TrxR and GPx activity during oxidative stress To investigate the mechanisms of Pycnogenol Ò on TrxR and GPx enzyme activity, the cells were exposed to oxidative stress in the form of H 2 O 2 . The cells were first incubated with Pycnogenol Ò for 48 h, followed by H 2 O 2 treatment at a concentration of 0.1 mm and incubation for an additional 48 h. Then the cells were harvested and homogenized and the enzyme activity was measured. Inhibition of enzyme activity of Pycno- genol Ò in control cells is evident for both TrxR and GPx (Fig. 4). The activity of TrxR was highly increased after exposure to oxidative stress compared to the control as shown in Fig. 4A. However, the increase was reversible for TrxR after addition of 25 lgÆmL )1 Pycnogenol Ò , declining to the basal level. GPx, on the other hand, barely responded to treatment with hydrogen peroxide, and Pycnogenol Ò did not lower GPx activity in the presence of hydrogen perox- ide (Fig. 4B). Fig. 3. Determination of ROS production. ROS production was detected using CM-H 2 DCFDA after 8 h treatment with 10 lgÆmL )1 Pycnogenol Ò alone or in combination with 7.5 lM selenite (Se), 500 l M Se-methyl-seleno-L-cysteine (SeMC), 750 lM tert-butyl hydroperoxide (tert-BuOOH) or 40 l M rotenone (positive control). Error bars represent the standard error based on three independent experiments. The Wilcoxon matched-pair test was used to compare effects between control ⁄ treatment experimental set-ups: **P < 0.01; ***P < 0.001. A B Fig. 4. TrxR and GPx activity after treatment with Pycnogenol Ò in combination with hydrogen peroxide. (A) TrxR activity and (B) GPx activity in cell homogenates pre-treated with 25 lgÆmL )1 Pycnoge- nol Ò for 24 h, followed by treatment with or without 0.1 mM H 2 O 2 for an additional 48 h. Black bars indicate the addition of 0.1 mM H 2 O 2 . Error bars represent the standard error based on three inde- pendent experiments. The Wilcoxon matched-pair test was used to compare effects between control ⁄ treatment experimental set-ups: *P < 0.05; **P < 0.01; ***P < 0.001. V. Gandin et al. Effects of Pycnogenol Ò on redox systems FEBS Journal 276 (2009) 532–540 ª 2008 The Authors Journal compilation ª 2008 FEBS 535 Effect of Pycnogenol Ò on cellular TrxR and GPx activity after treatment with selenite As previously reported [27], both TrxR and GPx activ- ity were elevated after selenite treatment (Fig. 5). In contrast to GPx, TrxR was clearly affected by the addition of Pycnogenol Ò , while GPx remained high activity following selenite treatment even after addition of Pycnogenol Ò (Fig. 5B). TrxR1 protein levels and TrxR1/TrxR2 mRNA expression As the TrxR activity was remarkably upregulated by both selenite and hydrogen peroxide, with a reversible affect after treatment with Pycnogenol Ò , the protein and mRNA expression were investigated. TrxR1 protein lev- els decreased remarkably, corresponding to the drop in activity. A significant decrease in mRNA expression was seen for both enzymes after treatment with 5 lgÆmL )1 Pycnogenol Ò alone (Fig. 6). Moreover, the same pattern with a decrease, as in mRNA expression was seen for the activity when treated in combination with either sel- enite or tert-BuOOH. The effect on TrxR1 mRNA was nevertheless much more pronounced compared to TrxR2. Discussion Pycnogenol Ò has a wide variety of highly interesting effects, including anti-inflammatory properties, benefi- cial effects on the vascular system, and protection from UV radiation [2]. Furthermore, Pycnogenol Ò is an extremely efficient antioxidant, and it is likely that many of its effects may be explained by its antioxidant action. The antioxidant effects of Pycnogenol Ò are fur- ther supported by our finding that Pycnogenol Ò decreases the production of hydrogen peroxide when added in combination with compounds known to pro- duce ROS, including selenite (Fig. 3). Even though our findings are based on use of a lung carcinoma cell line, we strongly believe that the action of Pycnogenol Ò is a general mechanism and not only applicable in our model cell system, as the antioxidant effects of Pycno- genol Ò have been explored in other cell lines by us (data not shown) and also described by others [2,3]. The antioxidant effects of Pycnogenol Ò clearly resulted in a decrease in protein expression together with a reduction in the cellular activity of TrxR. The effect on GPx was much less pronounced, but still showed a slight inhibition. The decrease in enzyme activity could however not be explained by direct inhibition of TrxR, as in vitro experiments with pure TrxR1 and Pycnoge- nol Ò did not result in any inhibitory effect. Mammalian TrxRs are complex selenoenzymes with many physiolog- ical functions, including reduction of thioredoxin and other low molecular weight substances, and reduction and detoxification of hydroperoxides [9]. This reduction may be direct or by regeneration of antioxidants includ- ing Q10, vitamin C and lipoic acid [13,28]. Another important function is to reduce selenium compounds, thereby providing active selenide for the synthesis of selenoproteins [29,30]. Our data show that addition of hydrogen peroxide led to increased enzyme activity, indicating that TrxR is regulated by the redox state of the cell. Furthermore, hydrogen peroxide could in part prevent the effect of Pycnogenol Ò and thus partly restore the activity of TrxR. One possible mechanism explaining the effect of Pycnogenol Ò on the activity of TrxR is that Pycnogenol Ò is an extremely efficient anti- oxidant that changes the redox status of the cell, thus Fig. 5. TrxR and GPx activity after treatment with Pycnogenol Ò in combination with selenite. (A) TrxR activity and (B) GPx activity in U1285 cell homogenates treated with Pycnogenol Ò alone (25 lgÆmL )1 ) or in combination with 5 lM selenite for 48 h. Black bars indicate the addition of selenite. Error bars represent the stan- dard error based on three independent experiments. The Wilcoxon matched-pair test was used to compare effects between control ⁄ treatment experimental set-ups: *P < 0.05; **P < 0.01. Effects of Pycnogenol Ò on redox systems V. Gandin et al. 536 FEBS Journal 276 (2009) 532–540 ª 2008 The Authors Journal compilation ª 2008 FEBS removing a stimulus for the synthesis of TrxR. How- ever, Pycnogenol Ò did not suppress TrxR1 to sub-basal levels. As shown in the ROS production experiment, Pycnogenol Ò completely reversed the ROS formation induced by both selenite and tert-BuOOH. However, the activity of GPx was barely affected by the addition of hydrogen peroxide after 48 h, implying a different response to oxidative stress to that seen for TrxR. Both TrxR and GPx are selenoproteins that are known to be regulated by the selenium content in the cell, with selenium generating a maximum expression level for TrxR and GPx at around 1 lm. However, TrxR1 is more readily saturated than GPx in response to selenium supplementation [31], probably due to the higher ranking of TrxR1 in terms of the hierarchy of selenoenzymes [32]. Treatment of cells with an organic selenium compound, SeMC, did not cause any cell death even at very high concentrations. SeMC is a natural selenium compound that is cleaved by b-lyase to highly redox-active monomethyl selenol [33]. How- ever, in the absence of b-lyase, monomethyl selenol is not formed, explaining the low toxicity of SeMC in U1285 cells. Despite the low cytotoxicity, SeMC caused increased ROS production in the cells. By using an inorganic selenium compound such as selenite as an oxidative agent, the regulation of expression of the anti- oxidant selenoproteins becomes much more complex. We show here that regulation of TrxR appears to be primarily dependent on the redox state of the cell, rather than the selenium content. This is demonstrated by the effect of addition of Pycnogenol Ò after selenite treatment, which decreases the activity of TrxR to levels below those of untreated cells, even though sele- nite alone resulted in a higher response compared to treatment with hydrogen peroxide. This was not the case for GPx activity, which, although it was consider- ably elevated after selenite treatment, was not at all affected by addition of Pycnogenol Ò . These findings are interesting and unexpected, as both enzymes have strong antioxidant properties and are dependent on selenium bioavailability. The decreased enzyme activity and mRNA expression of TrxR caused by Pycnogenol Ò alone and in combination with oxidants also gives an insight in the complex regulation of TrxR. These observations indicate that there is a defined hierarchy at various regulatory levels. The antioxidant-response elements in the promoter region of TrxR, which are targeted by nuclear erythroid-2-related factor (Nrf2) [34], are probably one of the most important regulatory elements for TrxR. Although our data suggest an effect of Pycnogenol Ò primarily at the transcription level, effects on translation may also occur. One example is the strictly redox-regulated translation factor SBP2, which is required for incorporation of selenocycstein A B Fig. 6. Protein levels of TrxR1 and TrxR1 ⁄ TrxR2 mRNA expression in the U1285 cell line. (A) Western blot comparing TrxR1 protein levels after treatment of cells for 48 h with Pycnogenol Ò (25 lgÆmL )1 ). Poly- clonal TrxR1 antibodies are known to generate more than one band due to the existence of several protein isoforms. (B) TrxR1 (black bars) and TrxR2 (grey bars) mRNA levels after treatment with 5 or 10 lgÆmL )1 Pycnogenol Ò alone or in combination with 5 l M selenite or 250 lM tert-BuOOH for 48 h. Error bars represent the standard error based on three indepen- dent experiments. The Wilcoxon matched- pair test was used to compare effects between control ⁄ treatment experimental set-ups: *P < 0.05; **P < 0.01; ***P < 0.001. V. Gandin et al. Effects of Pycnogenol Ò on redox systems FEBS Journal 276 (2009) 532–540 ª 2008 The Authors Journal compilation ª 2008 FEBS 537 into selenoproteins. SBP2 is translocated to the nucleus when oxidized, resulting in translational inhibition of TrxR [8]. The less pronounced effects seen for TrxR2 may be explained by the localization of TrxR2 in the mitochondria, which may be slightly less affected than the cytosol under these conditions. As TrxR is induced in various pathological conditions, including tumours and inflammatory conditions [35], decreased activity mediated by a non-toxic agent such as Pycnogenol Ò would be beneficial, and may offer a mechanistic expla- nation for the effects of Pycnogenol Ò . Experimental procedures Chemicals 5,5¢-dithiobis(2-nitrobenzoic acid) (DTNB), guanidine HCl, sodium deoxycholate, sodium selenite, glutathione, GPx from bovine erythrocytes, insulin from bovine pancreas, and NADPH were all purchased from Sigma (St Louis, MO, USA). Baker’s yeast GR was obtained from Fluka (Buchs, Switzerland), Escherichia coli Trx1 was purchased from Pro- mega, and recombinant rat TrxR1 was generously provided by Elias Arne ´ r (Department of MBB, Karolinska Institutet, Stockholm, Sweden). Pycnogenol Ò was kindly provided by Horphag Research Ltd (Geneva, Switzerland). Se-methyl- seleno-l-cysteine (SeMC) was purchased from PharmaSe (Lubbock, TX, USA). Cell line The cells (U1285, a small-cell lung carcinoma cell line [36]) were cultured in RPMI-1640 medium with GlutaMAX-1 and 25 mm Hepes (Invitrogen, Paisley, UK), supplemented with 10% heat-inactivated fetal bovine serum. The cells were maintained in a humidified incubator with 5% CO 2 at 37 °C. The cells were cultured in the presence of indicated concentrations of Pycnogenol Ò for various time periods. The Pycnogenol Ò powder was solved in NaCl ⁄ P i containing 25 mm Hepes. The effect of Pycnogenol Ò was also studied in combination with selenite, SeMC, tert-butyl hydroper- oxide (tert-BuOOH) and H 2 O 2 . Selenite, SeMC or tert- BuOOH, was added together with Pycnogenol Ò , while H 2 O 2 was added after 48 h of Pycnogenol Ò pre-treatment. Preparation of cell homogenates Buffer (50 mm Tris ⁄ HCl pH 7.6 and 1 mm EDTA) was added to the cell pellet, after which it was kept on ice. The cells were sonicated three times for 15 seconds, followed by centrifugation at 25 200 g for 10 min at 2 °C. The super- natants were stored at )20 ° C for later enzyme activity stu- dies. The protein concentrations in the cell homogenates were determined by the Biuret method [37]. TrxR enzyme activity assay The activity of the TrxR enzyme was measured through a coupled reaction in the cell homogenates essentially as described previously [38]. From each homogenate, 200 lg proteins were incubated with 80 mm Hepes (pH 7.5), 0.9 mgÆmL )1 NADPH, 6 mm EDTA, 2 mgÆmL )1 insulin and 10 lm E. coli Trx at 37 °C for 20 min in a final volume of 120 lL. The reaction was terminated by addition of 500 lL DTNB (0.4 mgÆmL )1 ) with 6 m guanidine HCl in 0.2 m Tris ⁄ HCl pH 8.0. The absorbance at 412 nm was measured using a SpectraMax 250 (Molecular Devices, Sunnyvale, CA, USA) within 20 min. To determine the effects of Pycnoge- nol Ò on pure TrxR1, 10 nm recombinant rat TrxR1 was used instead of the homogenate, with the addition of only 5 lm E. coli Trx1. GR enzyme activity assay Determination of the GR enzyme activity was performed as for the TrxR1 enzyme activity except that insulin was replaced by 1 mm oxidized glutathione, and 10 lgof protein was incubated for each sample. GPx enzyme activity assay The GPx enzyme activity was measured through a coupled reaction as described previously [39]. A modified protocol was created to fit a 96-well plate. For each cell homoge- nate, 200 lg of protein was incubated for 3 min at 25 °C with 0.1 m Tris ⁄ HCl buffer (pH 7.6), 2 mm EDTA, 2 mm NaN 3 ,4mm glutathione, 10 units of GR and 0.8 mm NADPH in a total volume of 195 lL. After incubation, H 2 O 2 was added to a final concentration of 10 mm as substrate for the GPx, and the absorbance at 340 nm was recorded. Viability assay In order to investigate the susceptibility of the cells to treatment with the various compounds, a Cell Prolifera- tion Kit II (XTT) from Roche Diagnostics (Mannheim, Germany) was used. Estimated IC 50 values from these trials were used to decide an appropriate range of con- centrations for treatment in all experiments conducted. The viability assay was performed in 96-well flat-bot- tomed culture dishes with 100 lL medium ⁄ well at approximately 10% cell confluence. Cells were subjected to treatment for 48 h, followed by an incubation time of 4 h before measurement. Absorbance was measured at 490 nm (with a reference wavelength of 650 nm sub- tracted) using a SpectraMax 250. All samples were measured in triplicate, and the entire assay was repeated three times. Effects of Pycnogenol Ò on redox systems V. Gandin et al. 538 FEBS Journal 276 (2009) 532–540 ª 2008 The Authors Journal compilation ª 2008 FEBS ROS production Intracellular ROS production was detected using non- fluorescent compound 5(6)-chloromethyl-2¢,7¢-dichlorodihy- drofluorescein diacetate (CM-H 2 DCFDA) (Invitrogen). CM-H 2 DCFDA undergoes deacetylation by intracellular esterases, and the product quantitatively reacts with oxygen species inside the cell to produce the fluorescent dye 5(6)- carboxy-2¢,7¢-dichlorofluorescein (CM-DCF). Briefly, U1285 cells (10 4 per well) were grown for 24 h in a 96-well plate in RPMI-1640 without phenol red. Subsequently, the medium was removed and the cells were incubated for 45 min in the dark with 10 lm CM-DCFDA in NaCl ⁄ P i . Excess probe was washed out with NaCl ⁄ P i , and cells were incubated with the reported concentrations of tested com- pounds for 8 h. The increase in fluorescence was deter- mined at wavelengths of 485 nm (excitation) and 527 nm (emission) on a SpectraMax 250. Western blotting Samples were analysed on 7.5% SDS–PAGE at 150 V, followed by semi-dry electroblotting to a nitrocellulose mem- brane for 1 h at 100 V. Membranes were probed with anti-TrxR1 (1 : 2000, Upstate, Lake Placid, NY, USA) and incubated for 1 h at room temperature. The membranes were further incubated with a horseradish peroxidase-conjugated secondary antibody (1 : 3000, DakoCytomation, Glostrup, Denmark). Bound antibodies were detected using a chemilu- minescence Western Lightning kit (PerkinElmer, Boston, MA, USA) according to the manufacturer’s instructions. Real-time PCR Total RNA was isolated using and RNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s proto- col. RNA quantification was performed using a Ribogreen RNA quantification kit (Molecular Probes, Eugene, OR, USA) according to the supplied ‘high range’ protocol with rRNA as the standard. Synthesis of cDNA was performed by reverse transcription of 2 lg of RNA using the Omni- script reverse transcription kit (Qiagen) with oligo(dT) 12–18 as primer (final concentration 0.1 lg Æ lL )1 ). Real-time quan- titative PCR was performed on a Bio-Rad ICycler (Bio-Rad, Hercules, CA, USA) with 20 ng of cDNA per reaction in triplicate on 96-well plates using Platinum SYBR Green qPCR super mix (Invitrogen). Determination of TrxR1 mRNA levels was performed as described previously [40]. TrxR2 mRNA was analyzed using the same program as for TrxR1 but with forward primer 5¢-TCAGAAGATCC TGGTGGACTCC-3¢ and reverse primer 5¢-TCGTGGG AACATTGTCGTAGTC-3¢, with concentration of 300 nm for each primer. The results were analysed using the 2 ÀDDC t method. The C T value cut-off was set at 32 cycles. The effi- ciency of the primer sets was 90 ± 5%. Acknowledgements This investigation was supported by Radiumhemmets Research Society, Horphag Research Ltd, the Swedish Medical Association, Karolinska Institutet Research Grants, and the Swedish Cancer Society. References 1 Nordberg J & Arne ´ r ES (2001) Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic Biol Med 31, 1287–1312. 2 Rohdewald P (2002) A review of the French maritime pine bark extract (Pycnogenol Ò ), a herbal medication with a diverse clinical pharmacology. 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Quantification of alternative mRNA species and identification of thioredoxin reduc- tase 1 isoforms in human tumor cells. Differentiation 75, 123–132. Effects of Pycnogenol Ò on redox systems V. Gandin. antioxi- dant effects of Pycnogenol Ò on redox enzymes, with emphasis on the regulation of TrxR. Results Effects of Pycnogenol Ò on cellular TrxR enzyme activity To investigate the effects of Pycnogenol Ò on