Role of thioredoxin-1 in apoptosis induction by α-tocopheryl succinate and TNF-related apoptosis-inducing ligand in mesothelioma cells

FEBS Letters 580 (2006) 2671–2676

Role of thioredoxin-1 in apoptosis induction by a-tocopheryl succinate and TNF-related apoptosis-inducing ligand in mesothelioma cells Ruth E. Freemana, Jiri Neuzila,b,* a

Apoptosis Research Group, Heart Foundation Research Centre, School of Medical Science, Griffith University, Southport, Qld 9716, Australia b Laboratory of Cell Signalling and Apoptosis, Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic Received 28 December 2005; revised 23 March 2006; accepted 5 April 2006 Available online 21 April 2006 Edited by Michael R. Bubb

Abstract Malignant mesothelioma (MM) is a fatal type of cancer. We studied the role of the redox-active protein thioredoxin-1 (Trx-1) in apoptosis induced in MM cells and their non-malignant counterparts (Met-5A) by a-tocopheryl succinate (aTOS) and TNF-related apoptosis-inducing ligand (TRAIL). MM cells were susceptible to a-TOS and less to TRAIL, while Met-5A cells were susceptible to TRAIL and resistant to aTOS. MM cells expressed very low level of the Trx-1 protein, which was high in Met-5A cells, while the level of Trx-1 mRNA was similar in all cell lines. Downregulation of Trx-1 further sensitised Met-5A cells to TRAIL but not to a-TOS. Our data suggest that the role of Trx-1 in apoptosis modulation is unrelated to its anti-oxidant properties.  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Apoptosis; Malignant mesothelioma; Thioredoxin1; a-Tocopheryl succinate; TNF-related apoptosis-inducing ligand

1. Introduction Malignant mesothelioma (MM) is a fatal neoplastic disease with poor prognosis and no reliable cure at present, which makes it a considerable challenge despite its relatively rare occurrence [1,2]. Etiology of MM is tightly linked to occupational hazard, in particular breathing asbestos fibres [3]. Although asbestos mining and processing is now banned in developed countries, the number of MM cases will increase due to its very long latency, and the frequency of new cases is not predicted to plateau before 2015 [4]. The reasons for low response of MM to treatment are not exactly known but may include a relatively high correlation of MM patients with SV40 positivity, although this premise is non-conclusive at present [5]. Another reason for resistance of MM to chemotherapy, often relying on induction of apoptosis in the target cancer cells, is a relatively poor response of MM cells to inducers of programmed cell death [6,7], which may be associated with SV40-dependent inactivation of p53 [8] or activation of the potent pro-survival mediator Akt [9]. * Corresponding author. Fax: +61 2 555 28804. E-mail address: j.neuzil@griffith.edu.au (J. Neuzil).

Abbreviations: ELISA, enzyme-linked immuno-sorbent assay; FCS, fetal calf serum; MM, malignant mesothelioma; Q-PCR, quantitative reversed-transcription PCR; ROS, reactive oxygen species; siRNA, short interfering RNA; a-TOS, a-tocopheryl succinate; TRAIL, TNFrelated apoptosis-inducing ligand; Trx-1, thioredoxin-1

Further, enhanced expression of the anti-apoptotic bcl-2 family proteins may also contribute to resistance of MM cells to apoptosis [10]. Novel approaches that would overcome resistance of MM cells to apoptosis and would be applicable to treatment of MM patients are therefore required. We have been studying the role of vitamin E analogues, epitomised by the redox silent a-tocopheryl succinate (a-TOS), as pro-apoptotic and anti-cancer agents [11] and have shown that they induce apoptosis selectively in malignant cells while being largely non-toxic to normal cells and tissues [12–15]. Importantly, too, we have shown that a-TOS suppresses human MM in two different mouse models [16,17]. The mechanism underlying apoptosis induced by a-TOS includes induction of the intrinsic apoptotic pathways [18–20], which also contribute to sensitisation of MM cells to the selective immunological apoptogen TNF-related apoptosis-inducing ligand (TRAIL) [14,21], similarly as shown for other pharmacological inducers of apoptosis in MM cells [22]. The selective apoptogenic effect of a-TOS towards MM cells can be ascribed to high levels of reactive oxygen species (ROS) generated in the cells as a response to the vitamin E analogue, which was not observed in non-malignant mesothelial cells [17]. This paradigm was shown for apoptosis induction by a-TOS in other cells [15,18,23,24]. In fact, generation of ROS appears to be obligatory for malignant cells to undergo apoptosis when challenged with a-TOS, since their efficient scavenging by antioxidant proteins, such as superoxide dismutase (SOD), as well as by both small anti-oxidants has proven anti-apoptotic [17,25]. Of anti-oxidant enzymes, thioredoxin-1 (Trx-1) has been suggested to play a diverse role in cancer cells. Not only does it act as a redox-active protein with anti-oxidant activity [26] and redox modulatory effector of mediators of signalling pathways [27], but it also regulates pathways, such as MAP kinase signalling, in a redox-independent manner [28]. In this communication, we studied the role of Trx-1 in apoptosis induction of MM cells and their non-malignant counterparts exposed to aTOS whose induction of apoptosis requires ROS, and to TRAIL that induces apoptosis via a redox-independent mechanism [25]. We show here that, surprisingly, Trx-1 appears more important for TRAIL- than for a-TOS-induced programmed cell death in MM cells. 2. Materials and methods 2.1. Cell culture and treatment The MM-BI (biphasic), Meso-2 (sarcomatose) and Ist-Mes-2 (epithelioid) human MM cell lines were used [29]. The mesothelial cell line, Met-5A (ATTC, Rockville, MD, USA), was used as a non-

0014-5793/$32.00  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.04.019

2672 malignant control. The cells were cultured in DMEM supplemented with 100 units/ml penicillin, 100 lg/ml streptomycin, and 10% fetal calf serum. Cells were treated with human recombinant TRAIL prepared as described elsewhere [30] and a-TOS (Sigma, Castle Hill, NSW, Australia) at 30 ng/ml and 50 lM, respectively. a-TOS was dissolved in ethanol and diluted in complete DMEM to the final concentration, and was added to cells at 0.1% (v/v) of ethanol. Cells deficient in mtDNA (q0 phenotype) were prepared and cultured as described elsewhere [18]. 2.2. Apoptosis detection Apoptosis was quantified using the annexin V-FITC method, which detects phosphatidyl serine (PS) externalised in early phases of apoptosis [31]. Briefly, cells were treated with a-TOS or TRAIL. Floating and attached cells were collected, washed and re-suspended in the binding buffer containing 2 ll annexin V-FITC (both BD Pharmingen, North Ryde, NSW, Australia), incubated for 30 min at 4 C in the dark, supplemented with propidium iodide (PI), and analysed by flow cytometry (FACScalibur, BD Pharmingen). 2.3. Western blotting Cells were treated as indicated, lysed in whole cell lysis buffer, spun down, the supernatants mixed with the sample buffer, the protein denatured and electrophoresed in 12% polyacrylamide gels. Separated proteins were transferred to a nylon membrane and blocked with 5% skim milk in TBS buffer for 30 min at room temperature. Immuno-detection was performed overnight at 4 C in TBS buffer using anti-Trx-1 IgG (Fl-105) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-MnSOD IgG (Calbiochem), and with anti-actin IgG (I-19) (Santa Cruz) as a loading control. After incubation with HRP-conjugated secondary IgG (Santa Cruz), the blots were developed using the Supersignal Chemiluminescence Kit (Pierce Biotechnology, Rockford, IL, USA), and the membranes were scanned using the Universal Hood II and Quantity One software (both BioRad, Hercules, CA, USA). 2.4. Downregulation of Trx-1 using short-interfering RNA (siRNA) Two Trx-1 siRNA sequences (Proligo Primers and Probes, Lismore, NSW, Australia) were used. Duplex 1: sense 5 0 -GUC UUC AAC GGG UUC ACC GdTdT-3 0 , antisense 5 0 -CGG UGA ACC CGU UGA AGA CdTdT-3 0 , corresponding to the target sequence 5 0 -AAC CAG TTG CCA TCT GCG TGA-3 0 ; duplex 2: sense 5 0 CUG UCA GGA UGU UGC UUC AdTdT-3 0 , antisense 5 0 -UGA AGC AAC AUC CUG ACA GdTdT-3 0 , corresponding to the target sequence of 5 0 -GAC TGT CAG GAT GTT GCT TCA-3 0 . Nonsilencing (NS) siRNA (Qiagen, Doncaster, Vic., Australia) used was: sense 5 0 -UUC UCC GAA CGU GUC ACG UdTdT-3 0 , antisense 5 0 -UUC UCC GAA CGU GUC ACG UdTdT-3 0 . For treatment, cells were seeded in 6-well plates at a 7 · 104 and left overnight to recuperate. A solution was made up of 5 lg siRNA duplexes dissolved in 100 ll of cell culture medium plus 15 ll of OligofectAmine (Invitrogen, Mt. Waverley, Vic., Australia). The solution was incubated for 30 min at room temperature and added to each well containing 1.9 ml of cell medium. The cells were incubated for 6 h, after which the medium was removed and complete DMEM added. The cells were incubated for 48 h and then assessed by ELISA for Trx-1 protein level and used in experiments. 2.5. Trx-1 ELISA The enzyme-linked immuno-sorbent assay (ELISA) for Trx-1 was based on the use of the monoclonal ‘capture’ IgG 1B3 and the biotinylated monoclonal ‘light up’ IgG 2B1 [32]. Briefly, the unlabelled IgG 1B3 was diluted to the final concentration of 20 lg/ml and added to 96-well plates at 100 ll/well and allowed to attach. To determine the concentration of Trx-1 in samples, plates were incubated at 4 C overnight with individual samples, and this was followed by the antigen capture steps: 100 ll of the biotinylated IgG 2B1 (diluted 1:500) was added to each well, the plates were incubated at 37 C for 2 h, reacted with 100 ll of avidin–horseradish–peroxidase conjugate (BioRad) diluted 1:1000, and the colour was developed using 3,3 0 ,5,5 0 -tetramethylbenzidine. Absorbance was read in a plate reader set at 620 nm.

R.E. Freeman, J. Neuzil / FEBS Letters 580 (2006) 2671–2676 2.6. Thioredoxin reductase assay The method is based on thioredoxin reductase (TrxR)-mediated reduction of SH groups in the substrate, Trx, which is detected using DTNB, as described in detail in a previously published protocol [33]. The activity was expressed as nmol/min/mg protein of NADPH formed. 2.7. Quantitative real-time-polymerase chain reaction (Q-PCR) Q-PCR was used to assess levels of Trx-1 mRNA in MM and Met5A cells, essentially as reported elsewhere [17]. The primers used for Trx-1 were forward: 5 0 -CCT TTC TTT CAT TCC CTC TCT TGA A-3 0 , reverse: 5 0 -GCA ACA TCC TGA CAG TCA TCC A-3 0 ; for 18S-M primer: 5 0 -TTC GAG GCC CTG TAA TTG GA-3 0 , reverse: 5 0 -GCA GCA ACT TTA ATA TAC GCT ATT GG-3 0 . Total mRNA was extracted with Trizol (Sigma) and purified using RNeasy Mini Columns (Qiagen). cDNA was synthesised and the product quantified on the TaqMan real-time PCR instrument using the SYBRGreen master mix (Invitrogen). The results were then analysed using the computer software supplied with the PCR program. Before Q-PCR, the reaction was verified by standard RT-PCR followed by EtBr visualisation of the products on a 2% agarose gel. 2.8. Statistical analysis All experiments were conducted at least three times, and data are shown as means ± S.E. Where shown, statistical significance (P < 0.05) was evaluated using the Student’s t-test.

3. Results and discussion In this study we were interested in the potential role of Trx1, an antioxidant protein that regulates apoptosis in redoxdependent and -independent manners, in programmed cell death induced in MM cells by two different agents. One of them is a-TOS that causes generation of ROS as an early event in apoptosis induction, the other is TRAIL acting in an ROSindependent way [18,23]. For these studies, we used three MM cell lines of different phenotypic origin, i.e. the epithelioid IstMes-2, the sarcomatose Meso-2 and the mixed (biphasic) phenotype MM-B1 cell lines, and a non-malignant mesothelial cell line Met-5A. We first explored apoptosis induced by a-TOS and TRAIL. Fig. 1 shows a converse effect of the two inducers on apoptosis in the malignant and non-malignant cells. While Met-5A cells were more resistant to a-TOS than their malignant counterparts, TRAIL was surprisingly more apoptogenic for the non-malignant cells. a-TOS is known to induce apoptosis by triggering the intrinsic pathway(s) involving generation of ROS. We have shown that small as well as proteineous antioxidants, including MnSOD, suppress apoptosis induced by the vitamin E analogue, concomitant with lowering of ROS accumulation [15,18,24]. We thus studied here the role of the mitochondrial function and also of MnSOD expression. To do so, we prepared mtDNA-deficient (q0) cells by long-term incubation with sub-lethal doses of EtBr [18]. Our recent data show that q0 cells are more resistant to a-TOS compared to their parental counterparts [18,25]. This does not seem to be the case for MM-BI q0 cells, since they showed a similar level of apoptosis as did the parental MM-BI cells, and a similar pattern was also observed when the cells were exposed to TRAIL (Fig. 2A). We next inspected the level of expression of the MnSOD protein by Western blotting both in the individual cell lines as well as in the MM-BI q0 cells. Fig. 2B reveals that MnSOD is expressed at comparable levels in the non-malignant Met-5A

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Fig. 1. Apoptosis induction in non-malignant mesothelial and MM cells by a-TOS and TRAIL. Met-5A, MM-BI, Meso-2 and Ist-Mes-2 cells were seeded in 24-well plates and allowed to reach 60–70% confluency. The cells were exposed to 50 lM a-TOS (upper panel) and 30 ng/ml hrTRAIL (lower panel) for the periods indicated. The cells were then harvested and assessed for the level of apoptosis using the annexin V method. Data shown are mean values ± S.D. (n = 5). The asterisk indicates statistical difference (P < 0.05) in apoptosis in Met5A cells and the malignant cell lines.

Fig. 2. Deficiency in mtDNA does not protect MM cells from a-TOSinduced apoptosis. (A) Parental MM-BI and MMB-I-q0 cells were seeded in 24-well plates, allowed to reach 60–70% confluency, and exposed to a-TOS and TRAIL at concentrations and for periods indicated. The cells were then harvested and assessed for the level of apoptosis using the annexin V method. (B) Western blotting for expression of MnSOD (1-MM-BI, 2-MM-BI-q0) and (C) flow cytometric evaluation (MFI, mean fluorescence intensity) for expression of Trx-1 in MM-BI (Par, parental cells) and MM-BI-q0 cells. Data shown in (A) and (C) are mean values ± S.D. (n = 3), images in (B) are representative of three independent experiments.

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cells and the MM cell lines, and no appreciable change of its expression was found for the Meso-2 q0 cells. Since ROS have been strongly implicated in apoptosis induction by a-TOS, we assessed the cytosolic levels of Trx-1 in MM cells and the non-malignant Met-5A cells. Fig. 3 demonstrates a surprising paradigm, revealing similar level of Trx-1 expression on the level of mRNA (detected by both the conventional RT-PCR and by Q-PCR), while very high levels of the Trx-1 protein were found in Met-5A cells with all three malignant cell lines showing levels of Trx-1 close to the detection limit. This unexpected difference in Trx-1 protein expression between Met-5A cells and the MM cell lines was observed both using Western blotting and by the very sensitive ELISA assay. Moreover, this was confirmed by immunofluorescence microscopy of MM and Met-5A cells (not shown). This cannot be explained by secretion of Trx-1 by MM cells, since we found barely detectable levels of Trx-1 protein in both MM and Met5A cell-conditioned medium (not shown). This is a surprising finding and suggests that mRNA expression of Trx-1 does not necessarily correspond to the extent of the protein expression. Although, to the best of our knowledge, this paradigm has not been shown for Trx-1 before, it is known that not always high levels of mRNA expression reflect expression of the corresponding protein, as shown, for example, for 15-lipoxygenase, whose mRNA is high in hematopoietic precursors but is not translated before the cells mature into erythrocytes [34]. Notwithstanding, our data clearly show that the Trx-1 protein is highly expressed in the non-malignant Met-5A cells and is extremely low in all three MM cell lines, while levels of the MnSOD protein are comparable in all tested lines. Moreover, thioredoxin reductase, important for maintaining Trx-1 in its functional form, showed comparable activity in MM and Met5A cells (not shown). Since these cell lines represent mesothelioma cells of different phenotypic origin, it is likely that our results may be extrapolated to other MM cell lines as well. It is beyond the scope of this manuscript to investigate the reasons for the discrepancy in Trx-1 mRNA and protein expression in MM and Met-5A cells, but, in analogy to 15-lipoxygenase, it is possible that the Trx-1 mRNA is not translated efficiently in the de-differentiated MM cell lines. Because the non-malignant Met-5A and MM cells feature different expression of Trx-1 and since they are also differently susceptible to apoptosis initiation by a-TOS and TRAIL, we next studied the effect of modulation of the Trx-1 level on apoptosis upon exposure to the two inducers. We thus attempted to overexpress Trx-1 in MM cells. However, we did not succeed in increasing the levels of the Trx-1 protein in the transfectants, probably due to its strict regulation by the MM cells. It is possible that some cell types have a mechanism whereby controlling very tightly the cytosolic levels of Trx-1 protein or degrading the newly synthesised protein very efficiently. Notwithstanding, this interpretation is highly speculative at this stage and needs further investigations, which are beyond the cope of this manuscript. Not surprisingly thus, both the parental cells and the ‘transfectants’ responded to both apoptogens in a similar way (not shown). Conversely, we studied the effect of knocking down Trx-1. To do this, two different duplexes of Trx-1 siRNA were used to knock down Trx-1 protein in Met-5A and MM-BI cells. Of them, duplex 1 suppressed Trx-1 protein expression by 80% and duplex to by 60% in MM-BI cells, while in Met-5A cells, duplex 1 was inefficient and duplex 2 knocked down Trx-1

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Fig. 3. Trx-1 levels are dramatically different in the non-malignant mesothelial and MM cells. Met-5A, MM-BI, Meso-2 and Ist-Mes-2 cells were assessed for the expression of thioredoxin-1 on the level of mRNA using RT-PCR and Q-PCR (A) and on the level of protein using Western blotting (B) and the ELISA method (C), as detailed in Section 2. The inset in (C) shows details of Trx-1 protein expression (in ng/mg total protein) for MM-BI (1), Meso-2 (2) and Ist-Met-2 cells (3). In (B), Western blotting for b-Actin is shown as a protein loading control. Data shown are mean values ± S.D. (n = 3), images are representative of three independent experiments.

protein by 85% (Fig. 4A and B). The siRNA-pre-treated cells were then exposed to a-TOS and TRAIL. Trx-1 knock-down in Met5A cells did not sensitise them to a-TOS (Fig. 4C), while the cells were much more susceptible to TRAIL-induced apoptosis (Fig. 4D). On the other hand, knocking down Trx-1 in the malignant MM-BI cells had no effect on their susceptibility to apoptosis induced by a-TOS (Fig. 4E) or TRAIL (Fig. 4F). In this project, we studied the role of Trx-1 in apoptosis induction in MM and non-malignant mesothelial (Met-5A) cells exposed to the pharmacological apoptogen a-TOS and the immunological apoptogen TRAIL, both being selective for cancer cells [12,35]. We found, rather surprisingly, that (i) MM cells are resistant to TRAIL while Met-5A cells are susceptible to TRAIL apoptosis; (ii) Met-5A cells express very high and MM cells very low levels of Trx-1 protein, while expression of Trx-1 mRNA is comparable; (iii) MM cells are resistant to Trx-1 over-expression; and (iv) knocking down Trx-1 does not change susceptibility of MM cells to apoptosis but sensitises Met-5A cells to TRAIL killing. Kahlos et al. showed that cancer tissue from MM patients features higher expression of the Trx-1 protein than the nonmalignant surrounding pleura [36]. This differs from our data revealing very low levels of Trx-1 and almost 50-fold higher levels of the protein in the non-malignant Met-5A cells. At the same time, we found comparable levels of Trx-1 mRNA in both the malignant and non-malignant cells. It is possible that mRNA of MM cells contains a silencer that suppresses efficient translation. Since the MM cell lines are originally explanted from MM patients, they represent a valid cellular model of MM. Moreover, the MM cell lines used in this

study are of the sarcomatose, epithelioid and biphasic phenotype, while the Met-5A cells are SV40-immortalised cells derived from normal human pleura [29]. The fact that high level of expression of the Trx-1 protein in Met-5A cells is not due to their immortalisation with SV40 was documented by assessing for Trx1 expression in two unrelated, non-malignant cell lines, the rat left ventricular myocytes HL-1 and the human endothelial cells EAhy926. In both cases, the levels of the protein were comparable with those in Met-5A cells (not shown). The role of Trx-1 in cancer is unclear at present. While it has been suggested that high levels of expression of the redox-active protein may be positively associated with the metastatic potential of cancers and, in general, with cancer etiology, it is possible that high expression of Trx-1 may be anti-apoptotic since it may act as a reductant of ROS generated by cancer cells in response to a variety of anti-cancer/pro-apoptotic agents [37,38]. It has been established that Trx-1 also regulates apoptosis via a redox-independent manner, by modulating the ASK1 protein, a constituent of the MAP kinase cascade [28]. Our data suggest that in mesothelioma, the role of Trx-1 in regulation of apoptosis may be independent of its redox activity, since its modulation does not affect susceptibility of the cells to a-TOS. In fact, knocking down Trx1 in the TRAILsensitive Met-5A cells makes them even more susceptible to the immunological apoptogen. In conclusion, we suggest that Trx-1 may have little role of apoptosis in MM cells, while it may be anti-apoptotic for non-malignant mesothelial cells exposed to TRAIL, via its redox-unrelated properties.

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Fig. 4. Knocking down Trx-1 levels sensitises non-malignant mesothelial cells to TRAIL apoptosis. Met5A and MM-BI cells were seeded in 24-well plates and treated with Trx-1 siRNA (Duplex 1, Duplex 2, or NS siRNA) as detailed in Section 2, and the level of Trx-1 protein expression assessed by the ELISA method (A, Met5A cells; B, MM-BI cells). Met5A cells pre-treated with Duplex 2 siRNA (C and E) and MM-BI cells pretreated with Duplex 1 siRNA (D and F) were exposed to 50 lM a-TOS (C and D) or 30 ng/ml TRAIL (E and F) at concentrations and times shown, harvested, and assessed for apoptosis using the annexin V method. Data shown are mean values ± S.D. (n = 3). Acknowledgements: We thank K. Tonissen for providing the Trx-1 plasmid, L. Andera for supplying hrTRAIL, J. DiTrapani and C. Cahill for providing antibodies for the ELISA assay and for hrTrx-1, K. Ashton for designing the Trx-1 mRNA primers, and A. Perkins, E. Swettenham, K. Venardos, and R. Lymbury for technical assistance. This work was supported in part by grants from the Dust Diseases Board of Australia and the Australian Research Council to J.N., and by project no. AV0Z50520514 awarded by the Academy of Sciences of the Czech Republic, and is a part of post-graduate project of R.E.F.

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