The Relationship of Thioredoxin-1 and Cisplatin Resistance: Its Impact on ROS and Oxidative Metabolism in Lung Cancer Cells

Published OnlineFirst January 16, 2012; DOI: 10.1158/1535-7163.MCT-11-0599

Molecular Cancer Therapeutic Discovery Therapeutics

Medhi Wangpaichitr1, Elizabeth J. Sullivan3, George Theodoropoulos1, Chunjing Wu1, Min You2, Lynn G. Feun2, Theodore J. Lampidis3, Macus T. Kuo4, and Niramol Savaraj1,2

Abstract Elimination of cisplatin-resistant lung cancer cells remains a major obstacle. We have shown that cisplatin- resistant tumors have higher reactive oxygen species (ROS) levels and can be exploited for targeted therapy. Here, we show that increased secretion of the antioxidant thioredoxin-1 (TRX1) resulted in lowered intracellular TRX1 and contributed to higher ROS in cisplatin-resistant tumors in vivo and in vitro. By reconstituting TRX1 protein in cisplatin-resistant cells, we increased sensitivity to cisplatin but decreased sensitivity to elesclomol (ROS inducer). Conversely, decreased TRX1 protein in parental cells reduced the sensitivity to cisplatin but increased sensitivity to elesclomol. Cisplatin-resistant cells had increased endogenous oxygen consumption and mitochondrial activity but decreased lactic acid production. They also exhibited higher levels of argininosuccinate synthetase (ASS) and fumarase mRNA, which contributed to oxidative metabolism (OXMET) when compared with parental cells. Restoring intracellular TRX1 protein in cisplatin-resistant cells resulted in lowering ASS and fumarase mRNAs, which in turn sensitized them to arginine deprivation. Interestingly, cisplatin-resistant cells also had signiﬁcantly higher basal levels of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Overexpressing TRX1 lowered ACC and FAS proteins expressions in cisplatin-resistant cells. Chemical inhibition and short interfering RNA of ACC resulted in signiﬁcant cell death in cisplatin-resistant compared with parental cells. Conversely, TRX1 overexpressed cisplatin-resistant cells resisted 5-(tetradecyloxy)-2-furoic acid (TOFA)-induced death. Collectively, lowering TRX1 expression through increased secretion leads cisplatin-resistant cells to higher ROS production and increased dependency on OXMET. These changes raise an intriguing therapeutic potential for future therapy in cisplatin-resistant lung cancer. Mol Cancer Ther; 11(3); 604–15. 2012 AACR.

Introduction molecular pathways (1), making cisplatin resistance diffi- Cisplatin is a widely used therapeutic agent in the treat- cult to overcome. Thus far, no drugs can reverse cisplatin ment of several types of solid tumors including lung cancer resistance or selectively kill cisplatin-resistant cells. (both small cell and non–small cell), head and neck, and We have previously discovered that elevated reactive ovarian cancer. The majority of patients with cancer initially oxygen species (ROS) are found in cisplatin-resistant cell respond to cisplatin treatment; however, development of lines (2); however, the question remains as to why this drug resistance is expected resulting in disease progression. occurs. A number of investigators have shown that cis- Mechanisms of resistance are complex and involvemultiple platin can inhibit thioredoxin reductase1 (TrxR1), a thioredoxin-1 (TRX1) reducing enzyme, which leads to increased ROS, resulting in further damage of DNA and Authors' Affiliations: 1Department of Veterans Affairs, South Florida VA subsequent cell death (3, 4). To avoid cell death caused by Foundation; 2Department of Medicine, Hematology/Oncology, University cisplatin, cells adapt to survive at high ROS levels and use 3 of Miami Miller School of Medicine; Department of Molecular Cell and less TrxR1/TRX1 but upregulate other antioxidant sys- Developmental Biology, University of Miami, Miami, Florida; and 4Depart- ment of Molecular Pathology, MD Anderson Cancer Center, Houston, tems (5). We have reported lower TRX1 in a panel of Texas cisplatin-resistant lung cancer cells (2). However, it is not Note: Supplementary material for this article is available at Molecular known whether decreased TRX1 protein was due to Cancer Therapeutics Online (http://mct.aacrjournals.org/). transcriptional downregulation or increased secretion. G. Theodoropoulos and E.J. Sullivan contributed equally to this study. M.T. Increased TRX1 secretion has been reported by others as Kuo and N. Savaraj contributed equally to this study. a result of cellular stress and after cisplatin treatment (6– Corresponding Author: Niramol Savaraj, Miami VA Healthcare System, 8). We investigated whether decreased TRX1 found in 1201 NW 16th street (111K), Miami, FL 33125. Phone: 305-5753143; Fax: cisplatin-resistant cells is regulated at the mRNA or the 305-5753375; E-mail: [email protected] protein level and report our results herein. doi: 10.1158/1535-7163.MCT-11-0599 TRX1, a disulfide-reducing dithiol enzyme, is an impor- 2012 American Association for Cancer Research. tant antioxidant protein that facilitates the reduction of

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other enzymes (9). TRX1 also interacts with certain tran- Assay of intracellular ROS/H2O2 scription factors, which are known to be redox regulated As previously described (2), cells were collected and via the dithiol–disulfide exchange reaction, thereby alter- intracellular H2O2 was measured by incubating with 10 ing their DNA-binding capacity (10, 11). Interestingly, it mmol/L of acetyl-penta-fluorobenzenesulfonyl fluoresce- has been reported recently that TRX1 is involved in in (APFB; EMD) at 37C for 30 minutes in the dark. Then, adipogenesis in fibroblast cells (12). This study showed the cells were washed once with PBS and centrifuged to that downregulation of TRX1 can lead to increased lipo- remove impermeable reagents. Cells were resuspended in genesis. Thus, it is possible that lower TRX1 found in 500 mL of PBS and analyzed in a fluorometer, FLUOstar cisplatin-resistant cells may have an impact on the fatty OPTIMA, BMG Labtech (excitation at 485 nm and emis- acid synthesis pathway and other pathways that are sion at 520 nm). involved in oxidative metabolism (OXMET). In this report, we present evidence that decreased TRX1 Assay of mitochondrial membrane potential expression in cisplatin-resistant lung cancer cells is an Cells were collected and incubated with 50 nmol/L of important mediator of ROS and reprograms lung cancer tetramethylrhodamine ethyl ester (TMRE; Invitrogen) at cells to become more reliant on OXMET. To our knowl- 37C for 30 minutes in the dark. Then, the cells were edge, this is the first report that shows that decreased washed once with PBS and centrifuged to remove imper- TRX1 in cisplatin-resistant lung cancer can result in meable reagents. Cells were resuspended in 500 mL of PBS increased ROS and alteration in tumor metabolism. These and analyzed in the Accuri Flow Cytometer (excitation at biochemical changes can be used as a target for future 544 nm and emission at 590 nm) for the mitochondrial treatment of cisplatin-resistant cells. membrane potential (MMP).

Materials and Methods Determination of the concentration of TRX1 in the Cell lines and reagents culture medium or in the plasma SCLC1 was derived from the bone marrow of a patient The concentration of TRX1 was determined by sand- with small cell lung carcinoma (SCLC). SR2 is the cisplatin- wich ELISA with 2 TRX1 antibodies. Tetramethylbenzi- resistant variant derived from SCLC1 that was generated dine was used as coloring agent (Chromogen). The by intermittent exposure to cisplatin. Non–small cell lung strength of coloring was proportional to the quantities of carcinoma (NSCLC) was established from metastatic ade- human TRX1. Briefly, 100 mL of samples (medium or nocarcinoma to the brain, and small cell is a cisplatin- plasma), standards, or reagent blank were incubated with resistant variant derived from NSCLC by intermittent 96-well precoated plate for 60 minutes at 37 C (IBL, Co., exposure to cisplatin. These cell lines have been previously Ltd). The precoated plate was washed vigorously with characterized (13–16). Note that SR2 exhibits 20-fold resis- wash buffer 7 times. One hundred microliters of labeled tances to cisplatin and NSCLC exhibits 7-fold resistance antibody solution was added to each well and incubated to both cisplatin and carboplatin. Elesclomol was kindly for 30 minutes at 37 C. The precoated plate was then provided by Synta Pharmaceuticals. The 5-(tetradecyloxy)- washed with wash buffer 9 times. One hundred micro- 2-furoic acid (TOFA) was purchased from Sigma. liters of tetramethylbenzidine was added to each well and incubated for 30 minutes at room temperature in the dark. Growth inhibition and cytotoxicity assay After 30 minutes, stop solution was added into each well, Cells were seeded in 24-well dishes and treated with and then subjected to ELISA plate reader at 450 nm against various concentrations of elesclomol or cisplatin for 72 reagent blank. TRX1 concentration was quantified against hours as described previously (13, 17). At 72 hours, the the standard curve and normalized with the cell number. culture mediums as well as the trypsinized cells were collected and this admixture was centrifuged at 400 g for Assay of TrxR activity 5 minutes. The cell pellet was resuspended in 1 mL of A TrxRkit (Cayman Chemical) was usedto measuretotal Hank’s buffer and assayed for live cells and death cells cellular TrxR as previously described (2). Briefly, cells were using trypan blue exclusion method. seeded at 4 105 and cell lysate was prepared by sonication using the conditions recommended by the manufacturer. Western blot analysis Total TrxR activity was detected by measuring the reduc- Cells were seeded at 1 105/mL onto 100-mm dishes, tion of 5,50-Dithio-bis(2-nitrobenzoic acid) with NADPH treated, collected, and lysed. Total protein was separated to thionitrobenzoate by UV spectrophotometer at 405 nm. on an SDS-PAGE, transferred onto a nitrocellulose membrane (Amersham Biosciences), and immunoblotted with RNA interference experiments ATP citrate lyase (ACL), acetyl-CoA carboxylase (ACC), A total of 8 105 cells were seeded in a 60 mm petri dish fatty acid synthase (FAS; Cell Signaling Inc.), TRX1 (BD and incubated for 24 hours. INTERFERin transfection Bioscience), TRX2 (Santa Cruz), citrate synthase (Sigma), reagent (Polyplus) was then used to transfect following or actin (Sigma; refs. 13, 17). The signal intensity was short interfering RNAs (siRNA; Dharmacon) siTRXC1 measured with a molecular imager Chemidoc system (GAAAAGUAUUCCAACGUGA), siTRXC2 (GGACG- with Quality One software (Bio-Rad). CUGCAGGUGAUAAA), siACC (SMARTpool siRNA,

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NM_198836), and siCONTROL (UAGCGACUAAACAC- Statistical analysis AUCAA; ref. 17). All statistical analyses were conducted from 3 separate measurements using the 2-tailed t test, and the results TRX1 overexpression experiment were expressed as mean SD. A P value of less than 0.05 TRX1-cDNA with restriction sites SgfI and Mlul was was considered as statistically significant. cloned in to the pCMV6 expression vector (Origene). The plasmid DNA was transfected into cultured cells (8 105) Results by Fugene 6 (Roche). After 24 hours, 0.5 mg/mL of Decreased TRX1 in cisplatin-resistant cells is due to neomycin (G418) was added to the culture media for increased secretion þ transfection selection. Two clonal populations (SR2TRX C1 To confirm that cisplatin-resistant cells have higher TRXþC2 and SR2 ) were selected and maintained in media basal levels of ROS, we assayed for H2O2 production containing 0.2 mg/mL of neomycin. per cell in cisplatin-resistant versus parental cells (Fig. 1A). Our previous results also showed that TRX1 was lower Assay for lactate production in cisplatin-resistant cells and most likely contributes to The lactate assay was carried out with a kit (Biovision). higher ROS levels. TRX has 2 functional isoforms; TRX1 Following the manufacture’s instruction, 200 mL/well of localizedinthecytoplasm and TRX2in the mitochondria. In media was filter through 10-kD molecular weight spin filter. this report, we have clarified that the TRX1 isoforms were Fifty microliters of supernatant were then added to reaction decreased whereas TRX2 showed no changes in all lung mix that contained lactate assay buffer, probe, enzyme mix, cancer cell lines tested (Fig. 1B). We then investigated and incubated for 30 minutes. Lactate levels were measur- whether decreased TRX1 protein was due to a decrease in ed by ELISA plate reader at 570 nm against reagent blank transcription by evaluating the TRX1 mRNA using RT- (media alone) and normalized with the cell number. PCR.TRX1mRNA levels weresimilarbetweenthe parental and cisplatin-resistant cells (Fig. 1C), whereas their activ- Qualitative real-time PCR ities were significantly lower (Fig. 1D). Thus, our data Qualitative real-time PCR (qRT-PCR) was carried out as suggest that the decreased TRX1 protein in cisplatin-resis- previously described (18). Briefly, 1mg of RNA was used tant cells is regulated post-transcription. Because it has for cDNA synthesis. The primers for qRT-PCR are been reported that TRX1 can be secreted from cells through designed with Beacon Designer for SYBR Green fluoro- the leaderless secretory pathway (19), it is possible that phore. Forty cycle amplification was used. The data were lower TRX1 found in cisplatin-resistant cells may be due to analyzed with iQ5 software from Bio-Rad. To calculate the the excessive secretion of this protein. We, therefore, com- DD relative mRNA level, we used the Ct method. The level pared the concentration of extracellular TRX1 in culture of mRNA was corrected with that of glyceraldehyde-3- media from the parental versus cisplatin-resistant cells. phosphate dehydrogenase (GAPDH). Results showed that TRX1 concentrations in culture media from cisplatin-resistant cells were significantly higher (Fig. Assay of oxygen consumption 1E). To further investigate whether decreased TRX1 protein Live cells were trypsinized and counted. Cells were expression and increased TRX secretion also occur in vivo, then resuspended in 1 mL of RPMI supplemented with 5% we chose SCLC1 (parental) and SR2 (cisplatin resistant) as FBS and 2 mg/mL of glucose. Each cell line was tested 3 or xenograft models in severe combined immunodeficient more independent times for a minimum of 6 readings per (SCID) mice and assayed for TRX1 in tumors via immuno- cell. Oxygen consumption readings were carried out as histochemical staining (Fig. 1F). Simultaneously, we suggested by the manufacturer (Yellow Springs Instru- assayed mouse serum for TRX1 (Table 1). SR2 tumors ments model 5300). clearly expressed less TRX1 intracellular protein consistent with the low intracellular expression we detected in vitro Immunohistochemistry staining (Fig. 1G). Mice with SR2 xenografts had higher TRX1 in Immunohistochemical staining was carried out accord- serum than mice with SCLC1 xenografts. These findings ing to routine methodology with some modification. We were consistent with the increased TRX1 secretion we used the target retrieval solution and the biotin block observed in culture (Fig. 1E). Taken together from the system (both from Dako) to enhance the staining. Samples in vitro and in vivo models, our findings suggest that were incubated overnight with primary antibody (1:200 in cisplatin-resistant cells have a lower intracellular TRX1 antibody dilution solution; Dako S3022) and washed. protein level that is not a transcriptional consequence, but Secondary antibody solution (Dako Link, biotinylated rather due to an increase in secretion, ultimately resulting antibody solution) was added for 25 minutes, washed, in higher ROS accumulation. and streptavidin conjugated to peroxidase (horseradish peroxidase) solution was added for 25 minutes. 3,30-Dia- Downregulation of TRX1 correlates with increased minobenzidine (DAB) chromogen (Dako) and then hema- ROS production, resistance to cisplatin, and sensitivity toxylin were used for staining. Section with only antibody to elesclomol in a cisplatin sensitive cell line solution was used as control. A modified protocol without To determine whether TRX1 correlates with ROS levels the rehydration steps was used for cells. and sensitivity to cisplatin as well as ROS-producing

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A B C TRX1 Actin Figure 1. Cisplatin-resistant (CR) 700 1 2 3 4 1 2 3 4 M lung cancer cells express lower 600 SCLC1 SR2 NSCLC SC levels of thioredoxin-1 (TRX1) 500 protein. A, ﬂuorometer analysis of 400 TRX1 CR CR H2O2 in various lung cancer cell lines 300 detected by APFB probe indicates 200 TRX2 100 (ROS production/cell) that cisplatin-resistant lung cancer Fluorescense intensity Actin cell lines expressed higher basal 0 SCLC1 SR2 NSCLC SC levels of ROS (Mean SD of 1:SCLC1, 2:SR2, 3:NSCLC, 4:SC, M:marker 3 experiments). B, immunoblot of TRX1 and TRX2 in lung cancer cell D TRX1 activity E lines showed that resistant variants * ** expressed lower levels of TRX1, 0.05 Measurement of TRX1 in culture medium ﬁ whereas no signi cant changes in 0.045 TRX2 occurred. Actin was used as a 1.6 0.04 loading control. C, RT-PCR of TRX1 1.4 ** 0.035 in lung cancer cell lines indicated that 1.2 all 4 cell lines have similar levels of 0.03 1

TRX1 mRNA expression. D, using mol/L/min/mL 0.025 µ 0.8 TrxR1 activity assay, cisplatin- 0.02 resistant cells have lower TRX1 0.6 * 0.015 activity than their parental cells. 0.4 TrxR TrxR 0.01 , P < 0.001; , P ¼ 0.003. E, the TRX1 ng/mL/cell 0.005 0.2 NSCLC SR2 SC concentration of extracellular TRX1 SCLC1 in culture medium. Cisplatin- 0 0 SCLC1 SR2 NSCLC SC resistant cells secreted greater Cell lines levels of TRX1 than their parental counterparts. , P ¼ 0.001; F In vivo G , P ¼ 0.002. F, immunohisto- SCLC1 SR2 chemistry of TRX1 in mouse In vitro xenograft tissue showed that SR2 SCLC1 SR2 has lower levels of TRX1 protein. G, immunocytochemistry of TRX1 in lung cancer cell cultures also showed decreased levels of TRX1 in cisplatin-resistant cells (200). SC, small cell. 100 µm 100 µm

100 µm 100 µm

agent (elesclomol), we inhibited TRX1 expression in We have previously shown that cisplatin-resistant cells SCLC1 using 2 different siRNA (SCLCsiTRXC1 and that have high ROS levels are sensitive to elesclomol (2). SCLCsiTRXC2). TRX1 protein was successfully knocked We anticipate that TRX1 knockdown cells that have high down (about 80%) in the transfected cells, SCLCsiTRXC1 ROS should be more sensitive to elesclomol. Indeed, we and SCLCsiTRXC2 (Fig. 2A, i and ii) and higher levels of have found that both TRX1 knocked down clones ROS were detected relative to SCLC1 (Fig. 2B, i and ii). (SCLC1siTRXC1 and SCLC1siTRXC2) were more sensitive to Nevertheless, the levels of ROS in TRX1-knocked down elesclomol (Fig. 2D, i and ii). At 30 nmol/L of elesclomol, cells were still lower than in SR2 cells. It is conceivable 70% of SCLC1 cells were still viable whereas only 35% of that mitochondrial ROS may also contribute to high ROS the knocked down cells survived. These data suggest that in SR2. Because silencing TRX1 may affect TRX2, we TRX1 plays a role in determining elesclomol sensitivity studied TRX2 in SCLC1siTRXC1 and found only slightly via alteration in ROS levels. increased in TRX2 levels (Fig. 2A). The participation of TRX2 in ROS accumulation remains unclear. Overexpression of TRX1 results in decreased ROS Next, we evaluated the viability of these cells under production, increased sensitivity to cisplatin cisplatin treatment. Both siTRX clones were signiﬁcantly treatment, and resistance to elesclomol in more resistant to cisplatin (Fig. 2C, i and ii). At 0.5 mg/mL of cisplatin-resistant cells cisplatin, 50–60% of the transfected cells were still viable To further verify the role of TRX1 in cisplatin resistance whereas only 20% of the SCLC1 cells survived. We could in lung cancer cells, we overexpressed TRX1 protein in notreach the similar cisplatin-resistant levels as in SR2 cells. SR2, using the pCMV6 vector containing full-length TRX1 ThismaybeduetothefactthatwecannotknockdownTRX1 cDNA. We were able to generate 2 stably transfected cells þ þ down to the same levels as in SR2 and that other mechan- (SR2TRX C1 and SR2TRX C2) that have about 3- to 4-fold isms of resistance in SR2 may be present (see Discussion). increase in TRX1 protein expression when compared with

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Table 1. The concentration of extracellular phosphorylation was used more in SR2, we measured MMP with TMRE staining. Higher MMP correlates to more TRX1 in mouse serum active oxidative phosphorylation (20). MMP was significantly higher in SR2 cells (Fig. 4C) than SCLC1, suggesting 3 Name Tumor volume, mm TRX1, ng/mL that cisplatin-resistant cells have more highly active Nontumor NA <0.62 mitochondria that can also contribute to increased ROS SCLC1 270 3.86 0.26 production. Higher mitochondrial activity implies that SR2 248 4.78 0.46 cisplatin-resistant cells may use more OXMET and less glycolytic flux. To consider this possibility, we assayed for NOTE: Mice-bearing SR2 xenografts were found to have the lactate production, which is a known indicator of higher levels of TRX1 in the serum. The value (ng/mL) repre- glycolytic metabolism. We found lower amounts of lactic P ¼ sents the average of 3 mice per group. ( 0.03; SCLC1 vs. acid (nmol/L/well/cell) in media from cisplatin-resistant SR2). cells (Fig. 4D). Thus, our initial findings suggest that cisplatin-resistant cells have higher mitochondrial activity and rely more on OXMET instead of glycolysis. SR2 (Fig. 3A, i and ii). These transfectants containing increased levels of TRX1 were used to study ROS pro- Cisplatin-resistant cells preferentially used OXMET, duction and sensitivity to cisplatin. Results showed that which was partially reversed upon overexpression of ROS production was significantly reduced in both TRX1- TRX1 overexpressed clones (Fig. 3B, i and ii) whereas cisplatin We further validated the involvement of specific sensitivity was increased (Fig. 3C, i and ii). At 2.5 mg/mL OXMET components as well as the impact of TRX1 on of cisplatin, 55% of SR2 cells were still viable whereas TRXþC1 TRXþC2 cisplatin-resistant lung cancer metabolism. To investigate about 30% and 40% of SR2 and SR2 sur- the dependence of cisplatin-resistant cells on OXMET and vived, respectively. Next, we analyzed the sensitivity of TRX1 as an important factor in this metabolic switching, both clones to the ROS-producing agent, elesclomol. As we studied key proteins in the tricarboxylic acid (TCA) predicted, elesclomol had a lesser antitumor effect in cycle, the urea cycle, and fatty acid synthesis using our cell TRX1-overexpressed cells than in SR2 cells. At 10 lines model. Citrate synthase is the first rate-determining nmol/L of elesclomol, only 45% of SR2 survived whereas TRXþC1 TRXþC2 enzyme in the TCA cycle (21, 22), whereas fumarase in SR2 and SR2 about 80% of the cells were participates in maintaining the equilibrium between suc- still viable (Fig. 3D, i and ii). Our data clearly indicate that cinate and fumarate. This equilibrium impacts the func- TRX1 levels have a major impact on high ROS levels seen tionality of complex II in the electron transport chain (21). in cisplatin-resistant cells and that manipulation of ROS We found no significant changes in citrate synthase pro- levels through TRX1 expression can alter cisplatin and tein in all 4 cell lines (data not shown); however, fumarase elesclomol sensitivity. Although further work in this area mRNA was increased in SR2 (1.8-fold) and attenuated in is needed, TRX1 may be used as a marker in the future þ þ TRX1-transfected cells, SR2TRX C1 and SR2TRX C2 (Fig. selection of patients for cisplatin and/or elesclomol 5A). Fumarase hydrates fumarate to malate, an important treatment. intermediate in the TCA cycle; however, the major source of fumarate is from the urea cycle (23). Hence, we inves- Cisplatin-resistant cells consume more oxygen and tigated 2 important enzymes in the urea cycle, arginino- have more mitochondrial activity succinate synthetase (ASS) and argininosuccinate lyase Although we have shown that low intracellular TRX1 (ASL), which generate arginine and yield fumarate as by- levels in cisplatin-resistant cells contribute to higher ROS product. Interestingly, SCLC1 had a negligible amount of levels, it is also possible that these cisplatin-resistant cells endogenous ASS mRNA (Fig. 5B) whereas SR2 had a have highly active mitochondria that are known to con- relatively robust expression (30-fold increase). Both TRX1 tribute to elevated ROS. To verify this, we first analyzed the overexpressed transfectants showed a decrease in ASS amount of oxygen consumed in SCLC1 versus SR2 cells. mRNA (Fig. 5B). ASL expression was not significantly SCLC1 consumed 0.6 nmol/L of O2/cell/mL of media, different in these 4 cell culture models, so it was not whereas SR2 consumed 2.4 nmol/L of O2/cell/mL of evaluated further (data not shown). These results imply media in the same time period (Fig. 4A). These data that SCLC1 will require an exogenous arginine supply for indicate that cisplatin-resistant cells use more oxygen their growth whereas SR2 should survive better in argi- (aerobic) than parental cells. In addition, under hypoxic nine-free media. To confirm the functional role of ASS, we conditions (0.5% O2), cell growth was restricted more in compared the growth sensitivity of these 4 cell lines to cisplatin-resistant cells than in parental cells (data not arginine-free media supplemented with citrulline, as an shown). This observation led us to assess mitochondrial ASS substrate. SCLC1 could not withstand arginine dep- activity through mitochondrial staining (MitoTracker). rivation due to lack of ASS expression, and hence, only SR2 had more intense fluorescence than SCLC1, which is 30% cells were viable after 48 hours in arginine-free indicative of higher mitochondrial activity (Fig. 4B and media. In contrast, 80% of SR2 were still viable. Impor- Supplementary Fig. S1). To further show that oxidative tantly, overexpression of TRX1 suppressed ASS in SR2,

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A scramble siTRXC1 B (i) SCLC1 SCLC1 SR2

TRX-1 (i) (ii) 700 700

TRX-2 600 600 * ** 500 500

400 400 Actin 300 300

200 200 (ROS production/cell) (ROS production/cell) Fluorescense intensity le Fluorescense intensity 100 100 scramb siTRXC2 C1 CLC1 0 0 (ii) SCL S SR2 SCLC1scramble SCLC1siTRXC1 SR2 SCLC1scramble SCLC1siTRXC2 SR2 TRX-1

Actin

Growth inhibitory assay

CD120 120 (i) SCLC1 (i) 100 * SCLC1 100 siTRXC1 siTRXC1 SR2 80 SR2 80 * ** ** 60 60 40 % Viable

% Viable 40 20 20 SCLC1 +Scramble SCLC1 SCLC1+siTRXC1 SR2 SR2 SCLC1 +Scramble SCLC1 SCLC1+siTRXC1 0 0 0 10 30 0 0.25 0.5 Elesclomol (nmol/L) Cisplatin (µg/mL) (ii) (ii) 120 120 Scramble Scramble siTRXC2 100 *** 100 siTRXC2 SR2 SR2 80 80 **** *** 60 **** 60 % Viable%

% Viable 40 40

20 20 SR2 SCLC1 +Scramble SCLC1+siTRXC2 SR2 SCLC1 +Scramble SCLC1+siTRXC2 0 0 0 10 30 0 0.25 0.5 Cisplatin (µg/mL) Elesclomol (nmol/L)

Figure 2. Downregulation of TRX1 results in increased ROS production, sensitivity to elesclomol, and resistance to cisplatin in a SCLC1 cell line. We selected SCLC1 that had the highest amount of TRX1, whereas its cisplatin-resistant variant (SR2) had the lowest amount of TRX1 to do the study. A, i and ii, immunoblot of TRX1 and TRX2 in SCLC1scramble (control), SR2, SCLCsiTRXC1, and SCLCsiTRXC2 cells. Two different siRNAs were able to downregulate TRX1 by 80% at 48 hours posttransfection, whereas no effect occurred in TRX2. Actin was used as a loading control. B, i and ii, downregulation of TRX1 resulted in signiﬁcant ROS production. , P ¼ 0.02; , P ¼ 0.035. C, i and ii, growth inhibitory effect of cisplatin for 72 hours showed that downregulation of TRX1 in SCLC1 cells resulted in resistance to cisplatin treatment. , P ¼ 0.02; , P ¼ 0.001; , P < 0.05; , P ¼ 0.04. D, i and ii, growth inhibitory effect of elesclomol for 72 hours showed that downregulation of TRX1inSCLC1resultsinincreasedsensitivityto elesclomol treatment. , P ¼ 0.01; , P ¼ 0.003; , P ¼ 0.12; , P ¼ 0.04. Mean SD of 3 experiments.

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A B (i) SCLC1 SR2 SR2 TRX+C1

TRX1 (i) (ii) * ** 700 700

600 600 Actin 500 500 400 400 300 300

SCLC1 SR2 SR2 TRX+C2 (ii) 200 200 (ROS production/cell)(ROS Fluorescense intensity 100 100

TRX1 Fluorescense intensity (ROS production/cell) 0 0 SCLC1 SR2 SR2 TRX+C1 SCLC1 SR2 SR2TRX+C2

Actin

Growth inhibitory assay CD (i) (i) 120 120 SR2 SR2TRX+C1 SR2 100 * 100 * SR2TRX+C1 ** 80 ** 80

60 60 % Viable % Viable 40 40

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0 0 0 1 2.5 0 5 10 (ii) Cisplatin (µg/mL) (ii) Elesclomol (nmol/L) 120 120 SR2 SR2 SR2TRX+C2 100 100 TRX+C2 *** *** SR2 **** 80 80 **** 60 60

% Viable 40 % Viable 40

20 20

0 0 0510 012.5 Cisplatin (µg/mL) Elesclomol (nmol/L)

Figure 3. Overexpression of TRX1 results in decreased ROS production, resistance to elesclomol, and sensitivity to cisplatin in the SR2 cell line. A, þ þ i and ii, immunoblot of TRX1 in SCLC1, SR2, SR2TRX C1,andSR2TRX C2 cells. Both TRX1-overexpressing clones expressed about 3- to 4-fold increase in TRX protein when compared with SR2. Actin was used as a loading control. B, i and ii, overexpression of TRX1 resulted in decreased ROS production in SR2 cell lines. , P ¼ 0.002; , P ¼ 0.01. C, i and ii, growth inhibitory effect of cisplatin for 72 hours showed that overexpression of TRX1 in SR2 resulted in increased sensitivity to cisplatin treatment. , P ¼ 0.001; P ¼ 0.03; , P < 0.05; , P < 0.05. D, i and ii, growth inhibitory effect of elesclomol for 72 hours indicated that overexpression of TRX1 in SR2 resulted in resistance to elesclomol treatment. , P ¼ 0.007; , P ¼ 0.006; , P ¼ 0.008; , P ¼ 0.004. Mean SD of 3 experiments.

which in turn sensitized them to arginine deprivation dria. Recently, it has been reported that overexpression of (Fig. 5C). the Txnip protein, an inhibitor of TRX1, can lead to Fatty acid synthesis also contributes to OXMET by adipogenesis (12). Thus, it is likely that the relatively providing carbon skeleton to b-oxidation in the mitochon- lower TRX1 expression found in SR2 may promote fatty

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Figure 4. Cisplatin-resistant cells Oxygen consumption B Mitochondria activity consume more oxygen and have A higher mitochondrial activity than 250 parental cells. A, SCLC1 and SR2 800 * were assayed for baseline oxygen 200 consumption. Live cells were 0.6 nmol/L/mL/cell 600 counted and placed in an oxygen SCLC1 150 SR2 chamber. The amount of oxygen 400 consumed per cell in 10 minutes 100 was measured. The rate of 200 oxygen consumption 2.4 nmol/L/mL/cell intensity per cell Oxygen (nmol/L/mL) 50 (O2 nmol/L/mL/cell/min) was 4 times 0 higher in SR2 cells than SCLC1 cells. Mitochondrial fluorescense SCLC1 SR2 B, SCLC1 and SR2 were incubated 0 with 100 nmol/L of MitoTracker. Bar 35302520151050 graph represents the relative Time (min) fluorescent units per cell via C D fluorometer plate reader. SR2 cells have a higher number of active MMP Lactate production mitochondria than SCLC1. 0.018 , P < 0.001. C, flow analysis of MMP * 0.016 in SCLC1 versus SR2, using 600 50 nmol/L of TMRE. SR2 has SCLC1 0.014 significantly higher levels of MMP. 0.012 , P < 0.001. D, media from the 400 * 0.01 SCLC1 versus SR2 cells was SR2 0.008 collected and used in a 0.006 Counts 200 chromatogenic assay to Lactate amount 0.004 (nmol/L/well/cell) measure amounts of lactic acid 0.002 (nmol/L/well/cell). SR2 cells 0 0 produced less lactic acid than SCLC1 SR2 101 102 103 104 105 106 107.2 cisplatin-resistant cells. Mean SD FL2-A of 3 experiments; , P < 0.05.

þ þ acid synthesis. To investigate this concept, we assayed 3 SR2TRX C1 and SR2TRX C2 cells from TOFA-induced cell important enzymes in fatty acid synthesis ACL, ACC, and death at all doses with about only 30% to 40% cell death at FAS. All 3 protein levels were higher in SR2 cells than the highest dose (10 mg/mL), which were 2-fold less than in SCLC1 (Fig. 5D); ACL (3-fold), ACC (15-fold), and SR2. Importantly, knockdown of ACC by about 90% using FAS (5-fold) increased (Fig. 5E). TRX1 overexpression siRNA (SR2siACC; Fig. 6B) resulted in significant cell death þ (SR2TRX C1) significantly decreased the expression of fatty (55%), whereas the control SR2 cells with scramble acid synthesis proteins. These data support the notion that sequence can survive with only 6% cell death (Fig. 6C). cisplatin-resistant cells rely on OXMET more than their Thus, our data strongly suggest that SR2 relies on fatty parental cells. Importantly, the overexpression of TRX1 acid synthesis for survival and decreased TRX1 impacts protein opposes OXMET by downregulating enzymes fatty acid metabolism dependency. involved in the urea cycle and fatty acid synthesis. Sim- þ ilarly, SR2TRX C2 also exhibited a decrease in ACC and FAS when compared with SR2 (data not shown). Discussion We previously reported that cisplatin-resistant cells Fatty acid synthesis is important for survival of have more basal ROS levels than parental cell counter- cisplatin-resistant cells parts. Furthermore, increasing ROS using elesclomol in The earlier data strongly suggest that SR2 depends on these cisplatin-resistant cells can push them beyond their fatty acid synthesis pathway for survival as SR2 expressed tolerance limit, which ultimately leads to cell death (2). 15-fold more ACC and 5-fold more FAS than SCLC1. To Elesclomol is an investigational drug that chelates copper further verify that fatty acid synthesis plays a vital role on (Cu) outside the cells and enters as elesclomol-Cu(II), þ þ SR2 survival, first, we treated SR2, SR2TRX C1, SR2TRX C2, generating ROS via redox cycling of Cu(II) to Cu(I) inside and SCLC1 with TOFA, an allosteric inhibitor of ACC that mitochondria (25, 26). This drug has entered into both blocks the synthesis of malonyl CoA (24) and then mea- phase I and II clinical study, and thus far has been very sured for cell death (Fig. 6A). TOFA induced cell death in a well tolerated (27, 28). dose responsive manner in all cells with SR2 showing Here, we showed that lower TRX1 levels were found in the highest sensitivity to TOFA, with 30% cell death at all cisplatin-resistant cells tested and this is primarily due 2.5 mg/mL and 70% at 10 mg/mL, which was 6- to 7-fold to the increase in secretion. High TRX1 levels were found higher than SCLC1. TRX1 overexpression rescued in the media as well as in the serum of the SR2 mouse

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A B C Figure 5. Cisplatin-resistant cells 120 2.5 35 SR2 preferentially used OXMET, which FH ASS partly reversed upon

SR2 30 100 TRX+C2 TRX+C2 TRX+C1

2 TRX+C1 overexpression of TRX1. A and B,

25 SR2 SR2 80 SR2 relative mRNA levels of fumarase SR2 SR2 1.5 20 SR2TRX+C2 and ASS. Total RNAs extracted TRX+C1 60 SCLC1 SR2 SCLC1 15 from these cells were reverse 1 SR2 40 transcribed and subsequently 10 % Viability SR2TRX+C1 SCLC1 used as template for qRT-PCR. 0.5 SR2TRX+C2 5 20 GAPDH was used as internal SCLC1 mRNA relative expression mRNA relative 0 0 0 control. The results shown in the 0 1 2 graph were calculated with the Cell lines Days in arginine-deprived media DDCt method by setting the fumarase or ASS mRNA level of DE SCLC1 as 1. Relative abundance of 12,000 fumarase and ASS mRNA were TRX+C1

ACL SR2 10,000 higher in cisplatin-resistant cells

8,000 SR2 but decreased with TRX1 overexpression. FH, fumarase. C, 6,000 SCLC1 SR2 SR2TRX+C1 comparison of growth inhibition of þ SCLC1 TRX C1 ) 4,000 SCLC1, SR2, SR2 ,and 2 TRXþC2 TRX1 2,000 SR2 in arginine-free media supplement with citrulline. At 72 0 hours, only 30% of SCLC1 survive 40,000 ACC compared with 60% to 65% of 35,000 ACL TRX1-overexpressing clones, and

30,000 SR2 80% of SR2 were viable. D, 25,000 immunoblot of key enzymes (ACL, 20,000 TRX+C1 ACC, and FAS) in the fatty acid ACC 15,000

SR2 synthesis pathway. Cisplatin- 10,000 resistant cells had relative higher

5,000 SCLC1 levels of expression in all of these 0 FAS proteins than parental cells. 7,000 FAS Overexpression TRX1 protein can 6,000 SR2 partly suppress these fatty acid 5,000 synthesis enzymes. Actin was

Actin TRX+C1 used as a loading control. E, bar

Relative adjusted density (intensity x mm adjusted density (intensity Relative 4,000 graph indicates the relative 3,000 SR2 adjusted density of indicated 2,000 protein expressions in each cell SCLC1 1,000 line. SR2TRXþC2 yielded similar 0 results (data not shown). Cell lines

xenograft model. TRX1, a 12-kD protein, is a member of explained on the basis of ROS that is influenced by TRX1 the leaderless protein family and can be effluxed from the levels, it is unclear why TRX1 levels also influence cis- cells through the leaderless pathway (6, 19). It has been platin sensitivity. It is noteworthy that SR2 has other shown that increased TRX1 secretion is found when cells mechanisms of resistance, such as decreased cisplatin are under stress (8, 29, 30). Increased plasma TRX1 levels uptake due to decreased copper transport (hCTR1), a are found in patients who received cisplatin treatment (31, known transporter of cisplatin into the cells and is influ- 32). It is very likely that these cisplatin-resistant cells enced by the redox system (33–35). Whether TRX1 has any excrete higher amounts of TRX1 due to continuous cel- impact on hCTR1 is not known and is currently under lular stress, which in turn results in lower intracellular investigation. accumulation of TRX1 and consequently increased ROS. TRX1 has been reported to be involved in tumor angio- To mimic low TRX1 levels observed in cisplatin-resis- genesis. Welsh and colleagues have shown that overex- tant cells, we used siRNA to decrease TRX1 levels. pression of TRX1 increased hypoxia-inducible factor 1a SCLCsiTRX cells generated more ROS and became resistant (HIF1a) whereas transfection of nonfunctional TRX1 to cisplatin but hypersensitive to elesclomol. Likewise, decreased HIF1a (36). Because HIF1a also regulates overexpressing TRX1 in cisplatin-resistant cells resulted enzymes involved in the glycolytic pathway, it is possible in decreased ROS and increased sensitivity to cisplatin, that decreased TRX1 found in cisplatin-resistant cells also but more resistant to elesclomol. Although the differential has an impact on glucose metabolism. In fact, we found sensitivity to elesclomol in these transfectants could be that basal HIF1a protein expression was lowered in all

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The Impact of TRX1 on ROS and Oxidative Metabolism

Figure 6. Downregulation of ACC enhances cell death in cisplatin- A B SR2scramble SR2siACC resistant cells. A, cells were treated by TOFA to inhibit ACC and cytotoxicity Cytotoxicity assay ACC was assessed. Data are shown as percentage of cell death as compared 80 with untreated samples for each cell 70 line. Overexpressing TRX1 protected Actin SCLC1 cisplatin-resistant cells against 60 TOFA-induced cell death. B, SR2TRX+C1 50 C downregulation of ACC by siRNA SR2TRX+C2 70 (only 1 nmol/L is used to minimize the SR2 siACC 40 SR2 off-target effect). Immunoblot of ACC 60 in SR2scramble and SRsiACC cells 30

% Cell death 50 showed more than 90% decrease in 20 ACC 48 hours posttransfection. Actin SCLC1 40 TRX+C2 was used as a loading control. C, 10 TRX+C1 comparison of cell death in 30 SR2 SR2 scramble siACC 0 SR2

SR2 and SR ; ACC % Cell death 20 knockdown resulted in signiﬁcant 12.5510 increase in cell death. D, a proposed TOFA (µg/mL) 10 SR2 model acquired resistance to cisplatin disrupted the redox system 0 through inhibition of TRX1 system Scrambled siACC (TrxR1/TRX1) causing TRX1 sequence sequence secretion. Decreased TRX1 resulted D in accumulation of cellular ROS. Acquired cisplatin TRX1 Cellular TRX1 secretion Further increased ROS using resistance stress (Decreased intercellularTRX1) elesclomol in these cisplatin-resistant system cells can push them beyond their ? ? tolerance limit, which ultimately leads to cell death. Decreased TRX1 levels Oxidative ROS may involve metabolic metabolism reprogramming by switching FA synthesis cisplatin-resistant cells from inhibitors ROS glycolysis toward OXMET. Inhibiting key metabolic enzymes in fatty acid Cell death synthesis pathway led to signiﬁcant Elesclomol cell death in cisplatin-resistant cells.

cisplatin-resistant cells tested (Supplementary Fig. S2). of ASS has been reported in multiple tumor types such as These cisplatin-resistant cells had a lower hexokinase-II melanoma, hepatocellular carcinoma, and SCLC (39–42). level (37) and a lower lactic acid production. Thus, it These tumors are all highly sensitive to arginine depri- seems that decreased TRX1 levels have a negative impact vation. In fact, arginine deprivation therapy using pegy- on the glycolytic pathway. We have further shown that lated arginine deiminase has been shown to have anti- lower TRX1 found in cisplatin-resistant cells had other tumor activity in tumors that do not express ASS consequences on the key enzyme (ASS) involved in the (18, 41, 43, 44). Thus, alteration of ASS expression by urea cycle. These cisplatin-resistant cells have signi- TRX1 could have future clinical implication for treatment. ficantly higher levels of ASS mRNA, whereas the parental During the generation of arginine, fumarate is pro- cells (high TRX1 and HIF1a) expressed negligible amount duced as a by-product. We have found that fumarase of ASS mRNA. Interestingly, we have previously reported was higher in SR2 cells and attenuated with TRX1 over- that HIF1a is a key transcription factor that negatively expression. Interestingly, it has been reported that fuma- regulates ASS (38). Here, we showed that cisplatin-resis- rase deficiency in renal cell carcinoma results in glycolytic tant cells that have lower TRX1 and decreased HIF1a addiction, HIF stabilization, and alteration in ROS (45), levels express increased levels of ASS. Consequently, which is similar to our parental cells. It is also noteworthy these cisplatin-resistant cells are able to synthesize argi- that the majority of renal cells also do not express ASS (39). nine from citrulline, and therefore, are able to survive in Increased ASS and fumarase expression in cisplatin-resis- arginine-free media whereas its parental cells could not. tant cells implies that more oxidative phosphorylation is In addition, overexpression of TRX1 in cisplatin-resistant taking place. This is also confirmed by increased oxygen cells resulted in lower ASS mRNA and less tolerance to consumption as well as higher mitochondrial activity arginine deprivation, which further confirms the role of found in cisplatin-resistant cells. To further support that TRX1 in ASS mRNA expression. This is an important levels of TRX1 influenced cisplatin-resistant cells to use finding that warrants further study as downregulation more OXMET, we showed that cisplatin-resistant cells

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upregulate the key enzymes (ACL, ACC, and FAS) in the Culture Collection, and our initial finding suggested that fatty acid synthesis pathway, whereas overexpression of the extent of decreased TRX1 as well as higher ROS TRX1 resulted in attenuated levels of these enzymes. accumulation correlates with the resistant levels. Overall, These data strongly suggest that TRX1 may play an these findings could have future implication for drug important role in lipid metabolism. Interestingly, Chut- development to selectively kill cisplatin-resistant cells kow and Lee have recently reported that inhibition of that have high ROS and low TRX1 levels. TRX1 via Txnip can lead to increase in lipogenesis in fibroblast cells (12). Taken together, these findings further Disclosure of Potential Conflicts of Interest support the role of TRX1 in tumor metabolism. Knock- No potential conflicts of interest were disclosed. down of ACC in SR2 resulted in significant cell death, which indicates that fatty acid synthesis is essential for Acknowledgments The authors thank Drs. Masazumi Nagai, Yumiko Wada, and Jim Sang cisplatin-resistant cell survival. This is further confirmed from Synta Pharmaceuticals for Elesclomol, reagents, and SCID mouse by the finding of significant differential sensitivity to ACC xenograft model. þ inhibitor (TOFA) in SR2 cells and in SR2TRX . Although the data presented here are limited to one pair of cisplatin- Grant Support This work was supported in part by J&E King Biomedical Research, resistant cell line with different clones of transfectants, our FL Department of Health (1KD08) to M. Wangpaichitr; VA Merit previous data has shown that 4 pairs and 2 additional Review and NIH/NCI (1R01 CA109578-01) to G. Theodoropoulos, primary cultures derive from patients who failed cisplatin C. Wu, M. You, L.G. Feun, and N. Savaraj; NIH/NCI (1R01CA149260) to M.T. Kuo. have higher ROS and low TRX1 levels (2). These cell lines The costs of publication of this article were defrayed in part by the also showed alteration in their tumor metabolism either payment of page charges. This article must therefore be hereby marked via TCA cycle or fatty acid or by both. The underlying advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mechanisms are being investigated and will be forthcom- ing. We have also established other cisplatin-resistant Received August 11, 2011; revised November 28, 2011; accepted January cells from H69 (SCLC) obtained from American Type 9, 2012; published OnlineFirst January 16, 2012.

References 1. Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of 13. Wangpaichitr M, Wu C, You M, Kuo MT, Feun L, Lampidis T, et al. resistance. Oncogene 2003;22:7265–79. Inhibition of mTOR restores cisplatin sensitivity through down-regu- 2. Wangpaichitr M, Wu C, You M, Maher JC, Dinh V, Feun LG, et al. N1, lation of growth and anti-apoptotic proteins. Eur J Pharmacol N3-Dimethyl-N1,N3-bis(phenylcarbonothioyl) propanedihydrazide 2008;591:124–7. (elesclomol) selectively kills cisplatin resistant lung cancer cells 14. Wu C, Wangpaichitr M, Feun L, Kuo MT, Robles C, Lampidis T, et al. through reactive oxygen species (ROS). Cancers (Basel) 2009;1:23–8. Overcoming cisplatin resistance by mTOR inhibitor in lung cancer. Mol 3. Saitoh M, Nishitoh H, Fujii M, Takeda K, Tobiume K, Sawada Y, et al. Cancer 2005;4:25. Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regu- 15. Savaraj N, Wu C, Wangpaichitr M, Kuo MT, Lampidis T, Robles C, et al. lating kinase (ASK) 1. EMBO J 1998;17:2596–606. Overexpression of mutated MRP4 in cisplatin resistant small cell lung 4. Sun Y, Rigas B. The thioredoxin system mediates redox-induced cell cancer cell line: collateral sensitivity to azidothymidine. Int J Oncol death in human colon cancer cells: implications for the mechanism of 2003;23:173–9. action of anticancer agents. Cancer Res 2008;68:8269–77. 16. Xu J, Tyan T, Cedrone E, Savaraj N, Wang N. Detection of 11q13 5. Witte AB, Anestal K, Jerremalm E, Ehrsson H, Arner ES. Inhibition of ampliﬁcation as the origin of a homogeneously staining region in small thioredoxin reductase but not of glutathione reductase by the major cell lung cancer by chromosome microdissection. Genes Chromo- classes of alkylating and platinum-containing anticancer compounds. somes Cancer 1996;17:172–8. Free Radic Biol Med 2005;39:696–703. 17. Wangpaichitr M, Savaraj N, Maher J, Kurtoglu M, Lampidis TJ. Intrin- 6. Rubartelli A, Bajetto A, Allavena G, Wollman E, Sitia R. Secretion of sically lower AKT, mammalian target of rapamycin, and hypoxia- thioredoxin by normal and neoplastic cells through a leaderless secre- inducible factor activity correlates with increased sensitivity to 2- tory pathway. J Biol Chem 1992;267:24161–4. deoxy-D-glucose under hypoxia in lung cancer cell lines. Mol Cancer 7. Rubartelli A, Bonifaci N, Sitia R. High rates of thioredoxin secretion cor- Ther 2008;7:1506–13. relate with growth arrest in hepatoma cells. Cancer Res 1995;55:675–80. 18. Feun L, You M, Wu CJ, Kuo MT, Wangpaichitr M, Spector S, et al. 8. Miyamoto S, Kawano H, Sakamoto T, Soejima H, Kajiwara I, Hokamaki Arginine deprivation as a targeted therapy for cancer. Curr Pharm Des J, et al. Increased plasma levels of thioredoxin in patients with coronary 2008;14:1049–57. spastic angina. Antioxid Redox Signal 2004;6:75–80. 19. Rubartelli A, Bajetto A, Bonifaci N, Di Blas E, Solito E, Sitia R. A novel 9. Powis G, Kirkpatrick DL. Thioredoxin signaling as a target for cancer way to get out of the cell. Cytotechnology 1993;11 Suppl 1:S37–40. therapy. Curr Opin Pharmacol 2007;7:392–7. 20. Huttemann M, Lee I, Pecinova A, Pecina P, Przyklenk K, Doan JW. 10. Wei SJ, Botero A, Hirota K, Bradbury CM, Markovina S, Laszlo A, et al. Regulation of oxidative phosphorylation, the mitochondrial membrane Thioredoxin nuclear translocation and interaction with redox factor-1 potential, and their role in human disease. J Bioenerg Biomembr activates the activator protein-1 transcription factor in response to 2008;40:445–56. ionizing radiation. Cancer Res 2000;60:6688–95. 21. McGilvery RW. Biochemistry: a functional approach. In: Dusseau J, 11. Ando K, Hirao S, Kabe Y, Ogura Y, Sato I, Yamaguchi Y, et al. A new editor. Chapter 11: The oxidation of coenzyme A. Philadelphia, PA: APE1/Ref-1-dependent pathway leading to reduction of NF-kappaB W.B. Saunders Company; 1970. p. 206–24. and AP-1, and activation of their DNA-binding activity. Nucleic Acids 22. Srere PA. An eclectic view of metabolic regulation: control of citrate Res 2008;36:4327–36. synthase activity. Adv Enzyme Regul 1970;9:221–33. 12. Chutkow WA, Lee RT. Thioredoxin regulates adipogenesis through 23. Ratner S, Anslow WP Jr, Petrack B. Biosynthesis of urea. VI. Enzymatic thioredoxin interacting protein (Txnip) protein stability. J Biol Chem cleavage of argininosuccinic acid to arginine and fumaric acid. J Biol 2011;286:29139–45. Chem 1953;204:115–25.

614 Mol Cancer Ther; 11(3) March 2012 Molecular Cancer Therapeutics

The Impact of TRX1 on ROS and Oxidative Metabolism

24. Halvorson DL, McCune SA. Inhibition of fatty acid synthesis in isolated 36. Welsh SJ, Bellamy WT, Briehl MM, Powis G. The redox protein adipocytes by 5-(tetradecyloxy)-2-furoic acid. Lipids 1984;19:851–6. thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha pro- 25. Nagai M, Vho N, Cu J, Wada Y. Elesclomol-Cu chelate selectively tein expression: Trx-1 overexpression results in increased vascular targets mitochondria to induce oxidative stress [abstract]. In: Proceed- endothelial growth factor production and enhanced tumor angiogen- ings of the 102nd Annual Meeting of the American Association for esis. Cancer Res 2002;62:5089–95. Cancer Research; 2011 Apr 2–6; Orlando, FL. Philadelphia (PA): AACR; 37. Sullivan EJ, Kurtoglu M, Wangpaichitr M, Savaraj N, Lampidis T. 2011. Abstract nr 2093. Metabolic changes associated with acquired cisplatin resistance 26. Toogood PL. Mitochondrial drugs. Curr Opin Chem Biol 2008;12:457– [abstract]. In: Proceedings of the 102nd Annual Meeting of the Amer- 63. ican Association for Cancer Research; 2011 Apr 2–6; Orlando, FL. 27. Berkenblit A, Eder JP Jr, Ryan DP, Seiden MV, Tatsuta N, Sherman Philadelphia (PA): AACR; 2011. Abstract nr 4087. ML, et al. Phase I clinical trial of STA-4783 in combination with 38. Tsai WB, Aiba I, Lee SY, Feun L, Savaraj N, Kuo MT. Resistance to paclitaxel in patients with refractory solid tumors. Clin Cancer Res arginine deiminase treatment in melanoma cells is associated with 2007;13:584–90. induced argininosuccinate synthetase expression involving c-Myc/ 28. O'Day S, Gonzalez R, Lawson D, Weber R, Hutchins L, Anderson C, HIF-1alpha/Sp4. Mol Cancer Ther 2009;8:3223–33. et al. Phase II, randomized, controlled, double-blinded trial of weekly 39. Yoon CY, Shim YJ, Kim EH, Lee JH, Won NH, Kim JH, et al. Renal cell elesclomol plus paclitaxel versus paclitaxel alone for stage IV meta- carcinoma does not express argininosuccinate synthetase and is static melanoma. J Clin Oncol 2009;27:5452–8. highly sensitive to arginine deprivation via arginine deiminase. Int J 29. Nakamura H, De Rosa S, Roederer M, Anderson MT, Dubs JG, Yodoi J, Cancer 2007;120:897–905. et al. Elevation of plasma thioredoxin levels in HIV-infected individuals. 40. Szlosarek PW, Klabatsa A, Pallaska A, Sheaff M, Smith P, Crook T, Int Immunol 1996;8:603–11. et al. In vivo loss of expression of argininosuccinate synthetase in 30. Nakamura H, Vaage J, Valen G, Padilla CA, Bjornstedt M, Holmgren A. malignant pleural mesothelioma is a biomarker for susceptibility to Measurements of plasma glutaredoxin and thioredoxin in healthy arginine depletion. Clin Cancer Res 2006;12:7126–31. volunteers and during open-heart surgery. Free Radic Biol Med 41. Ensor CM, Holtsberg FW, Bomalaski JS, Clark MA. Pegylated arginine 1998;24:1176–86. deiminase (ADI-SS PEG20,000 mw) inhibits human melanomas and 31. Sasada T, Nakamura H, Ueda S, Iwata S, Ueno M, Takabayashi A, hepatocellular carcinomas in vitro and in vivo. Cancer Res 2002; et al. Secretion of thioredoxin enhances cellular resistance to 62:5443–50. cis-diamminedichloroplatinum (II). Antioxid Redox Signal 2000;2: 42. Prieto V, Curley S, Ensor C, Holzberg F, Bomalaski J, Clak M. Incidence 695–705. and distribution of argininosuccinate synthetase deﬁciency in human 32. Sasada T, Nakamura H, Ueda S, Sato N, Kitaoka Y, Gon Y, et al. cancers: a method for identifying cancers sensitive to arginine dep- Possible involvement of thioredoxin reductase as well as thioredoxin in rivation by arginine deiminase. Proc Am Soc Clin Oncol 22: 2003 cellular sensitivity to cis-diamminedichloroplatinum (II). Free Radic Biol (suppl; abstr 968). Med 1999;27:504–14. 43. Savaraj N, You M, Wu C, Wangpaichitr M, Kuo MT, Feun LG. Arginine 33. Chen HH, Song IS, Hossain A, Choi MK, Yamane Y, Liang ZD, et al. deprivation, autophagy, apoptosis (AAA) for the treatment of melano- Elevated glutathione levels confer cellular sensitization to cisplatin ma. Curr Mol Med 2010;10:405–12. toxicity by up-regulation of copper transporter hCtr1. Mol Pharmacol 44. Ascierto PA, Scala S, Castello G, Daponte A, Simeone E, Ottaiano A, 2008;74:697–704. et al. Pegylated arginine deiminase treatment of patients with meta- 34. Kuo MT, Chen HH, Song IS, Savaraj N, Ishikawa T. The roles of copper static melanoma: results from phase I and II studies. J Clin Oncol transporters in cisplatin resistance. Cancer Metastasis Rev 2007;26: 2005;23:7660–8. 71–83. 45. Sudarshan S, Sourbier C, Kong HS, Block K, Valera Romero VA, Yang 35. Song IS, Savaraj N, Siddik ZH, Liu P, Wei Y, Wu CJ, et al. Role of human Y, et al. Fumarate hydratase deﬁciency in renal cancer induces gly- copper transporter Ctr1 in the transport of platinum-based antitumor colytic addiction and hypoxia-inducible transcription factor 1alpha agents in cisplatin-sensitive and cisplatin-resistant cells. Mol Cancer stabilization by glucose-dependent generation of reactive oxygen Ther 2004;3:1543–9. species. Mol Cell Biol 2009;29:4080–90.

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The Relationship of Thioredoxin-1 and Cisplatin Resistance: Its Impact on ROS and Oxidative Metabolism in Lung Cancer Cells

Medhi Wangpaichitr, Elizabeth J. Sullivan, George Theodoropoulos, et al.

Mol Cancer Ther 2012;11:604-615. Published OnlineFirst January 16, 2012.

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