Endogenous Thioredoxin Is Required for Redox Cycling of Anthracyclines and p53-dependent Apoptosis in Cancer Cells*

Apoptosis is a major mechanism of cancer cell destruction by chemotherapy and radiotherapy. The anthracycline class of antitumor drugs undergoes redox cycling in living cells producing increased amounts of reactive oxygen species and semiquinone radical, both of which can cause DNA damage, and consequently trigger apoptotic death of cancer cells. We show here that MCF-7 cells overexpressing thioredoxin (Trx) were more apoptotic in response to daunomycin. Trx overexpression in MCF-7 cells increased the generation of superoxide anion (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{{\dot{2}}}^{-}\) \end{document}) in anthracycline-treated cell extracts. Enhanced generation of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{{\dot{2}}}^{-}\) \end{document} in response to daunomycin inTrx-overexpressing MCF-7 cells was inhibited by diphenyleneiodonium chloride, a general NADPH reductase inhibitor, demonstrating that Trx provides reducing equivalents to a bioreductive enzyme for redox cycling of daunomycin. Additionally Trx increased p53-DNA binding and expression in response to anthracyclines. MCF-7 cells expressing mutant redox-inactive Trx showed decreased superoxide generation, apoptosis, and p53 protein and DNA binding. In addition, down-regulation of endogenous Trx expression by small interfering RNA resulted in decreased expression of caspase-7 and cleaved poly(ADP-ribose) polymerase expression in response to daunomycin. These results suggest that endogenous Trx is required for anthracycline-mediated apoptosis of breast cancer cells. Taken together, our data demonstrate a novel pro-oxidant and proapoptotic role of Trx in anthracycline-mediated apoptosis in anthracycline chemotherapy.

Thioredoxin (Trx) 2 is a low molecular mass protein (12 kDa) that is widely distributed; Trx is found within the cytoplasmic, membrane, extracellular, and mitochondrial cellular fractions (1, 2). The Trx system includes Trx, Trx reductase, and peroxiredoxins. Trx reductase is an efficient protein-disulfide reductase that uses NADPH as a source of reducing equivalents. Besides being an antioxidant itself (3,4), Trx also plays an important role in regulating the expression of other antioxidant genes such as manganese superoxide dismutase (5). Trx overexpression also enhances the expression of peroxiredoxin that could reduce perox-ides to molecular oxygen and H 2 O (6). Trx has been shown to regenerate oxidatively inactivated proteins (7,8). In addition to its role as an antioxidant protein, Trx has been shown to have growth promoting properties (9). In contrast, a recent study has demonstrated that overexpression of redox-active Trx could promote cell death via activation of caspase-8 (10). Additional studies have shown that Trx reductase is critical for cell death, and a Trx-dependent mechanism has been suggested (11). Recent studies also indicate that caspases, the executioner of cell death by apoptosis, could be activated by Trx due to its disulfide reducing properties (12). Caspases are rich in cysteine motifs that are required for their catalytic activity. Therefore, oxidation could inhibit caspase activity, which could be restored by the Trx system (12). Furthermore Trx also has been shown to promote p53-DNA binding due to its reducing actions on DNA-binding cysteine motifs on p53 (14). Taken together, accumulating evidence suggests that Trx is a multifunctional protein, which can participate in proliferation as well as cell death process. The antioxidative action of Trx could be due to its manganese superoxide dismutase inducing properties (5,15) as well as direct scavenging of hydroxyl radicals or singlet oxygen.
The anthracycline class of anticancer drugs such as doxorubicin or daunomycin has been shown to induce p53-dependent apoptosis in cancer cells (16,17). Additionally anthracyclines have also been shown to cause DNA damage, which increases p53 expression (18,19). p53 is a sequence-specific transcription factor, which can induce proapoptotic or suppress antiapoptotic genes in response to DNA damage or irreparable cell cycle arrest (20). Phosphorylation of p53 on the Ser 15 residue dissociates MDM2 and activates p53 as a transcription factor, which binds to various p53-dependent genes resulting in their activation or repression (20). While evaluating the protective effect of Trx in daunomycin-induced cytotoxicity we observed increased death of MCF-7 cells overexpressing Trx. Because Trx has been shown to protect against oxidative stress and daunomycin-mediated cytotoxicity has been shown to be mediated in part by reactive oxygen species (ROS), our observation was rather surprising and novel. Anthracyclines contain quinone moieties in their structure, which can undergo biochemical reduction by one or two electrons catalyzed by flavoenzymes in the cell using NADPH as an electron donor (21)(22)(23). This bioreductive process generates semiquinone radical with concomitant production of superoxide anion (O 2 . ). The semiquinone radical intercalates with the DNA resulting in DNA damage. The formation of O 2 . is the beginning of a cascade that generates hydrogen peroxide and hydroxyl radicals, generally referred to as reactive oxygen species (24). In addition to various bioreductive enzymes, low molecular weight protein or non-protein thiols may also take part in the redox cycling process (25).
In the present investigation we report that endogenous Trx is required for daunomycin-induced apoptosis of cancer cells. In addition, we also demonstrate that Trx enhances the apoptotic death of cancer cells in response to daunomycin due to enhanced redox cycling of anthracyclines. In contrast, cells that express redox-inactive Trx or transfected with Trx siRNA show resistance to apoptosis.
Cell Culture and Adenovirus Production-MCF-7 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 100 units of penicillin/streptomycin. MCF-7 clones expressing Trx (Trx9), dominant negative redox-inactive Trx (Serb4), and only vector (vector) were the generous contribution of Dr. Garth Powis (Arizona Cancer Center, Tucson, AZ) and have been described previously (26,27). MCF-7 clones were cultured in Dulbecco's modified Eagle's medium containing G418 (300 g/ml). A549 cells were obtained from ATCC and propagated in F12K medium. The AdenoX system was obtained from Stratagene Corp. (La Jolla, CA), and Trx or mutant Trx open reading frame (26) was cloned into pAdenoX vector. Recombinant virus was allowed to infect human embryonic kidney 293 cells for generation of viral particles. For transfection, MCF-7 cells were infected with ϳ1 ϫ 10 8 infectious units (per million cells), and after 48 h protein expression was determined using enzyme-linked immunosorbent assay.
RNA Interference-p53, Trx, and scrambled non-targeting siRNA were purchased from Dharmacon RNA Technologies (Lafayette, CO). For transfection, MCF-7 or A549 cells were seeded in 35-mm 2 dishes to obtain 20% confluency at the time of transfection. Xtreme siRNA transfection reagent (Roche Applied Science) was used to transfect siRNA to a final concentration of 100 nM. Inhibition of gene expression by siRNA was determined after 72 h by Western analysis.
Thioredoxin Activity Assay-The thioredoxin activity assay was performed as described by Holmgren and Bjornstedt (1). Briefly the reaction mixture contained NADPH (200 M), porcine insulin (80 M, Sigma), and bovine Trx reductase (0.1 M) in 0.05 M potassium phosphate buffer (pH 7.0) containing EDTA (1 mM) in a total volume of 0.5 ml. The reaction was started by addition of bovine Trx reductase (0.1 M). Trx activity was calculated as micromoles of NADPH oxidized per minute per milligram of protein at 25°C (Beckman DU800 spectrophotometer).
TUNEL Assay-Apoptotic cells were detected using an in situ cell death detection, peroxidase kit (Roche Applied Science). Apoptotic DNA strand breaks were identified by labeling 3Ј-OH termini with fluorescein-dUTP using terminal deoxynucleotidyltransferase according to the manufacturer's protocol. Cells were allowed to adhere overnight in chambered glass slides (Nunc) to a final density of 25,000 cells/well. Following treatment with the appropriate concentration of drugs medium was removed, and cells were washed twice with PBS containing 1% bovine serum albumin and fixed in 4% paraformaldehyde for 30 min. Cells were then permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice and washed twice with PBS containing 1% bovine serum albumin. The labeling reaction was performed using fluorescein isothiocyanate-labeled dUTP along with other nucleotides by terminal deoxynucleotidyltransferase for 60 min in the dark at 37°C in a humidified chamber. Then cells were washed with PBS containing 1% bovine serum albumin and mounted, and the incorporated fluorescein-dUTP was analyzed using a fluorescence microscope (Axiovert M200, Carl Zeiss).
Flow Cytometry-Cells were treated with drugs for 48 h. Floating cells were collected, and adherent cells were washed with PBS and trypsinized. Floating and adherent cells were pooled and centrifuged at 500 ϫ g for 3 min. Cells were washed again with PBS containing 1% fetal bovine serum, resuspended in 500 ml of PBS followed by fixing in 7.5 ml of ice-cold ethanol (70%), added dropwise while vortexing, and stored at Ϫ20°C overnight. After two washes with PBS containing 1% fetal bovine serum, cells were resuspended in the same buffer and stained with 10 mg/ml propidium iodide (Sigma) in the presence of 250 mg/ml RNase at 37°C for 30 min in the dark. Stained cells were analyzed using a Coulter Epics Elite ESP flow cytometer using an argon laser at 488 nm wavelength. Flow cytometric results were analyzed, and apoptosis was defined as the "sub G 1 " peak (6) using Multicycle software.
Western Blotting-Protein lysates were prepared using radioimmunoprecipitation assay buffer containing 5% sodium deoxycholate, 1% SDS, 1% Igepal in PBS with protease inhibitors, and protein concentration was determined using Bio-Rad protein assay reagent. Equal amounts of protein were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane (Hybond-ECL, Amersham Biosciences). The blot was treated with the appropriate dilutions of primary antibody and visualized using either Lumiglo (Cell Signaling Technology) or the ECL plus system (Amersham Biosciences) with the appropriate horseradish peroxidase-conjugated secondary antibody.
Determination Clonogenic Assay-Cells were trypsinized and seeded to a final density of 1 ϫ 10 6 viable cells/100-mm 2 dish and allowed to attach overnight. Cells were then treated with the appropriate concentration of drugs for 24 h, trypsinized, and seeded to a final density of 500,000 viable cells/100-mm 2 dish. Viable cells were determined using a Vicell counter (Beckman Coulter). After 14 days, the surviving colonies were washed in PBS, fixed in 70% ethanol, and stained using 0.1% crystal violet in 90% ethanol. Colonies containing a minimum of 30 cells were counted using the colony counting feature present in the Quantity One software from Bio-Rad. The assays were performed in triplicate, and the data were statistically analyzed using InStat version 2.01 software.
Nuclear Extract Preparation-Nuclear extract was prepared as described previously (29). Briefly cells were washed in ice-cold PBS and  in 2 ml of ice-cold PBS by centrifugation. Cell pellets were resuspended in 400 l of Buffer A (10 mM HEPES, pH 7.8, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 50 g/ml leupeptin and antipain by gentle pipetting. Cells were allowed to swell on ice for 15 min followed by addition of 25 ml of 10% Nonidet P-40 and vortexed at full speed for 10 s. The homogenate was centrifuged for 30 s at 14,000 rpm. The nuclear pellet was resuspended in buffer C (20 mM HEPES, pH 7.8, 0.42 M NaCl, 5 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride in 10% (v/v) glycerol), and tubes were rocked gently at 4°C for 30 min on a shaking platform. The extracts were then centrifuged at 14,000 rpm for 25 min, and the supernatant was saved as nuclear extract at Ϫ70°C for further experiments. Protein was quantified using the Bradford protein assay (Bio-Rad).
Electrophoretic Mobility Shift Assay (EMSA)-For the EMSA, the p53 consensus oligonucleotide was obtained from Genosys (5Ј-GGCAT-GTCCGGGCATGTCC-3Ј) and was end-labeled using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [␥-32 P]ATP (PerkinElmer Life Sciences) in 10ϫ kinase buffer supplied with the enzyme. Ten micrograms of nuclear protein were preincubated in 5 l of 5ϫ binding buffer (20% glycerol, 5 mM MgCl 2 , 5 mM EDTA, 5 mM dithiothreitol, 500 mM NaCl, 50 mM Tris⅐HCl, 0.4 mg/ml calf thymus DNA), 200 ng of anti-p53 polyclonal antibody 421 and 2 g of poly(dI-dC) for 15 min followed by binding with labeled oligonucleotide for 30 min. The nuclear protein was separated by electrophoresis using a 4% native polyacrylamide gel and 0.25ϫ Tris borate-EDTA as running buffer. Gels were dried and exposed to Kodak Biomax x-ray film overnight.
Thioredoxin Enzyme-linked Immunosorbent Assay-Cells were homogenized in 50 mM Tris⅐HCl (pH 7.5) containing 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 g/ml leupeptin, and 20 g/ml antipain. Lysates were microcentrifuged for 10 min at 14,000 rpm. The protein concentration in the supernatant was measured using the Bradford method (Bio-Rad) with bovine serum albumin as standard. Enzyme-linked immunosorbent assay was performed as described previously (30).
Statistical Analysis-All statistical analysis was performed using the InStat software program (versions 2.01 and 3.0). All experiments were repeated at least twice.

Increased Expression of Redox-active Trx Enhances Apoptosis in
Response to Daunomycin-To test whether Trx overexpression protects MCF-7 cells against daunomycin-mediated apoptosis, we treated vector, Trx9, or Serb4 cells (clones of MCF-7 cells) with daunomycin and determined apoptosis as described under "Experimental Procedures." First we determined apoptosis using TUNEL assay, which detects nicks in the DNA that are generated during DNA damage and apoptosis. We expected to find more TUNEL-positive nuclei in Serb4 cells compared with Trx9 cells in response to daunomycin because previous studies show that Trx could protect against the cytotoxic actions of other anticancer drugs such as cisplatin and bleomycin (31)(32)(33). However, to our surprise we observed a higher number of TUNEL-positive Trx9 cells (Fig. 1, A and B) in response to daunomycin. TUNEL assay detects nicks generated by drug-induced DNA damage and endonuclease activation during apoptosis. MCF-7 cells lack caspase-3 and do not undergo clas-sical DNA laddering during apoptosis. Therefore, we further determined apoptotic cells as the "sub-G 1 " population by flow cytometry by propidium iodide staining. Treatment of vector, Trx9 or Serb4 clones with daunomycin resulted in the appearance of apoptotic cells ( Fig. 1, C and D). Trx9 cells showed a higher percentage of apoptotic cells (23%) compared with vector or Serb4 cells (8%). These data also agree with our TUNEL data that show increased apoptosis of Trx9 cells in response to daunomycin. Trx activity was assayed in vector, Serb4, and Trx9 cells using insulin reduction assay as described under "Experimental Procedures" (Fig. 1E).
Decreased Clonogenic Survival of MCF-7 Cells Overexpressing Trx-After cytotoxic treatment, cells can survive DNA damage through various repair processes and may continue to propagate into colonies (34,35). Therefore we compared the clonogenic survival of Trx9, Serb4, or vector cells treated with daunomycin at concentrations as low as 0.025 M. A clonogenic assay is a more stringent assessment of chemosensitivity than TUNEL or sub-G 1 peak measurements (34,35). Trx9 cells treated with daunomycin formed significantly less colonies at the end of FIGURE 2. Enhanced clonogenic death of MCF-7 cells and A549 cells overexpressing redox-active Trx but not redox-inactive Trx. A, vector, Serb4, or Trx9 cells were trypsinized and seeded to a final density of 1 ϫ 10 6 viable cells/100-mm 2 dish and allowed to attach overnight. Cells were then treated with the appropriate concentration of drugs for 24 h, trypsinized, and seeded to a final density of 500,000 viable cells/100mm 2 dish. Viable cells were determined using a Vicell counter (Beckman Coulter). After 14 days, the surviving colonies were washed in PBS, fixed in 70% ethanol, and stained using 0.1% crystal violet in 90% ethanol. B, stable clones of A549 cells expressing Trx or redox-inactive Trx (dnTrx) were processed as described in A. 14 days, whereas vector cells treated with daunomycin formed several colonies ( Fig. 2A). Thus MCF-7 cells overexpressing Trx exhibited increased apoptosis and decreased clonogenic survival in the presence of daunomycin as compared with vector-transfected cells.
In addition to MCF-7 clones, we also generated Trx-or dnTrx-expressing clones in A549 cells to test whether the observations with MCF-7 cells could be reproduced in other cell types. As demonstrated in Fig. 2B, A549-vector only clones or A549-dnTrx clones showed several colonies at the end of 14 days, whereas A549-Trx clones did not show a significant number of colonies. These data demonstrate that either A549 cells or MCF-7 cells expressing a higher level of Trx undergo increased apoptosis and decreased clonogenic survival in response to daunomycin. Next to determine whether endogenous Trx contributes to apoptosis induced by anthracyclines, we used RNA interference to down-regulate Trx expression.
Silencing Trx Expression by siRNA Decreases Apoptosis in MCF-7 Cells as Well as in A549 Cells in Response to Daunomycin-The apoptosis experiments described above were done using Trx-or dnTrxoverexpressing clones of MCF-7 cells. Therefore, there is reason to believe that the data obtained could be specifically applicable to a specific clone of Trx or dnTrx of MCF-7 cells. In addition, it is not clear whether the effects that were observed are only related to the overexpression of Trx. Therefore, to understand the role of endogenous Trx in apoptosis induced by daunomycin, we down-regulated the level of Trx in MCF-7 cells using a siRNA approach. As shown in Fig. 3A, treatment of MCF-7 cells with Trx siRNA down-regulated the Trx protein level. The decrease was about 95% compared with cells transfected with nontargeting siRNA. Following siRNA transfection, cells were treated with daunomycin, and the levels of apoptotic markers were evaluated by Western analysis. As shown in Fig. 3B (top panel) and Recent studies have demonstrated that caspase-1 expression is upregulated in response to doxorubicin in a p53-dependent manner in MCF-7 cells (36,37). Because caspase-1 expression is proapoptotic and its expression is dependent on p53, we examined the level of caspase-1 in response to daunomycin treatment and the effect of silencing Trx on the expression of caspase-1. In addition, we also examined the expression of caspase-6 and cleaved caspase-8 expression in response to Trx silencing in daunomycin-treated MCF-7 cells. As demonstrated in Fig.  3, F and G, caspase-1 expression was significantly up-regulated in response to daunomycin in MCF-7 cells transfected with non-targeting siRNA. In contrast, cells transfected with Trx siRNA demonstrated significant inhibition of caspase-1 up-regulation in response to daunomy-  DECEMBER 2, 2005 • VOLUME 280 • NUMBER 48 cin. However, the level of caspase-6 remained unchanged in response to daunomycin (Fig. 3F, middle panel). Treatment of cells with daunomycin resulted in the appearance of cleaved caspase-8 product (Fig. 3F,  lower panel), but the cleaved caspase-8 level remained unchanged in Trx siRNA-treated cells compared with non-targeting siRNA-treated cells. Taken together, these data suggest that caspase-1 and caspase-7 levels are modulated by daunomycin treatment, and Trx plays an important role in regulation of expression of these caspases.

Thioredoxin Enhances Apoptosis
To determine whether the role played by Trx in MCF-7 cells could be reproduced in other cell lines, we transfected lung adenocarcinoma A549 cells with siRNA for Trx or a non-targeting siRNA sequence and treated these cells with daunomycin. As demonstrated in Fig. 4A, transfection of Trx siRNA significantly down-regulated Trx level in A549 cells. These cells were treated with daunomycin, and the level of p53 was determined by quantitative immunoblotting. As shown in Fig. 4, B and C, down-regulation of Trx protein levels by siRNA significantly lowered p53 protein level similar to that observed with MCF-7 cells. Taken together, these data demonstrate that endogenous Trx is required for daunomycin-induced apoptosis of cancer cells. Next we determined how p53 expression is modulated in the presence of higher level of redox-active Trx or in the absence of redox-active Trx.

MCF-7 Cells Expressing Higher Trx Activity Show Higher p53 Protein Levels in Response to
Daunomycin-Anticancer agents that induce DNA damage also induce p53-mediated apoptosis (38). In the event of DNA damage, p53 is activated by phosphorylation and binds to the consensus sequence of the DNA, resulting in the regulation of gene expression required for apoptosis or cell cycle arrest (39). Phosphorylation of p53 blocks MDM2 binding and thereby prevents degradation of p53 protein resulting in accumulation of p53 (40). To determine the role of Trx in daunomycin-induced p53 protein expression, we examined the level and the extent of phosphorylation of p53 in Trx9 cells. Treatment of cells with 1 M daunomycin increased p53 protein in all clones (Fig. 5A, upper panel); however, in Trx9 cells there was a 2-3-fold increase in p53 protein level compared with vector or Serb4 cells. Phosphorylation of p53 at Ser 15 has been shown to be crucial in activating p53 as a transcription factor (17). Conformational changes due to phos-  ). B, stable clones of MCF-7 cells expressing redox-active Trx or redox-inactive Trx (dnTrx) were generated by transfecting cells with pCMV-Trx or pCMV-dnTrx constructs and selecting clones using 800 g/ml G418. Expression of Trx was determined by Western analysis as described previously. C, MCF-7 clones were treated with 1 M daunomycin for 16 h followed by p53 Western analysis as described previously. Lower panel, ␤-actin of same blot. D, overexpression of Trx or dnTrx in MCF-7 cells using AdenoX (Adx) infection. MCF-7 cells were infected with AdenoX LacZ, AdenoX Trx, or AdenoX dnTrx, and Trx expression was determined using an enzyme-linked immunosorbent assay as described previously (30). The amount of Trx was expressed as picograms of Trx per milligram of protein. *, significantly higher than control at p Ͻ 0.05 level. E, MCF-7 cells were infected with adenovirus (AdenoX LacZ, AdenoX Trx, or AdenoX dnTrx) as described under "Experimental Procedures." After 48 h infected cells were treated with 1 M daunomycin for 16 h. Western analysis for p53 was performed as described under "Experimental Procedures." Lane 1, cells infected with AdenoX LacZ; lane 2, cells treated with AdenoX Trx; lane 3, cells treated with AdenoX dnTrx; lanes 4 -6, cells infected with AdenoX LacZ, AdenoX Trx, or AdenoX dnTrx and treated with 1 M daunomycin for 16 h. ␤-Actin is shown as loading control. F, MCF-7 cells were transfected with Trx or dnTrx vectors as described under "Experimental Procedures." Cells were treated with 1 M daunomycin after 48 h, and following 16 h of incubation with the drug Western analysis was performed for p53 and ␤-actin as described under "Experimental Procedures." phorylation are required for its DNA binding activity (41). Therefore, we next evaluated the phosphorylation state of p53 on Ser 15 in daunomycin-treated cells using phosphospecific antibodies (Ser 15 ). Trx9 cells exhibited a higher phospho-p53 (Ser 15 ) level in response to daunomycin compared with vector or Serb4 cells, suggesting that a higher level of Trx could be potentiating the apoptotic potential of daunomycin (Fig.  5A, lower panel).
To determine whether the results obtained with a specific clone overexpressing Trx could be reproduced in other clones of MCF-7 cells overexpressing Trx, we generated several clones of Trx and dnTrx as described under "Experimental Procedures" in MCF-7 cells as shown in Fig. 5B. These clones were treated with 1 M daunomycin for 24 h followed by detection of p53 expression using Western analysis. As shown in Fig. 5C MCF-7 clones Trx25 and Trx26 showed higher p53 expression in response to daunomycin. In contrast, dnTrx clones 1 and 4 did not show higher p53 expression compared with vector only clones or Trx clones. These studies show that the results observed in vector, Trx9, or Serb4 cells could be reproduced in other MCF-7 clones. Therefore, using different clones of MCF-7 cells or other cells (A549) we observed increased p53 expression in response to daunomycin in Trxoverexpressing cells.
Next we examined whether transient overexpression of Trx in MCF-7 cells could produce results similar to those obtained using stable clones because there is reason to believe that stable expression of a protein could be clone-specific. Additionally the physiological response of stable clones could be different from that of transient overexpression. We used adenovirus-mediated gene delivery as well as Lipofectamine-   DECEMBER 2, 2005 • VOLUME 280 • NUMBER 48  Fig. 5D, adenovirus infection increased the Trx or dnTrx level in MCF-7 cells. When these cells were treated with daunomycin increased p53 expression was observed in MCF-7 cells overexpressing redox-active Trx (Fig. 5E). In contrast, dnTrx-overexpressing cells demonstrated almost no induction of p53 (Fig. 5E). Additionally as shown in Fig. 5F, transient transfection also increased Trx or dnTrx levels in MCF-7 cells (data not shown). When these cells were treated with daunomycin we observed a similar pattern of p53 expression (Fig. 5F). Taken together, these data indicate that overexpression of Trx enhances p53 expression in response to daunomycin, whereas expression of redox-inactive Trx inhibits the expression of p53 in response to daunomycin. p53-DNA Binding Is Enhanced in Trx9 Cells in Response to Daunomycin-Because phosphorylation of p53 activates its DNA binding that results in the regulation of the expression of many different genes involved in apoptosis or DNA repair, we used EMSAs to measure p53 binding to the DNA in nuclear extracts of cells treated with or without daunomycin. As demonstrated in Fig. 6A, daunomycin-induced p53-DNA binding was several -fold higher in Trx9 cells than in vector or Serb4 cells. Increased DNA binding implies increased transcription of p53-dependent genes, which may lead to increased apoptosis. We also determined the p53-DNA binding in MCF-7 cells transiently overexpressing Trx or its mutant form. As shown in Fig. 6B, higher levels of p53-DNA binding were observed in cells expressing higher levels of Trx. These studies indicate that a higher Trx level could enhance the transcription of p53-dependent genes that could enhance apoptosis.

Thioredoxin Enhances Apoptosis
Silencing p53 Decreases Expression of Apoptotic Proteins Such as Active Caspase-7 or Cleaved PARP-Because daunomycin treatment in Trx siRNA-transfected cells resulted in decreased p53 accumulation and apoptosis, we sought to determine whether the observed decrease in apoptosis in daunomycin is dependent on the level of p53 accumulation. We used p53 siRNA to inhibit p53 expression in MCF-7 cells (Fig.  7, upper panel) and treated these cells with daunomycin. Transfection of MCF-7 cells with p53 siRNA resulted in about 90% decrease in the expression of p53 (Fig. 7A, upper panel, and Fig. 7B). We also observed a decrease in the level of caspase-7 (Fig. 7A, middle panel, and Fig. 7C) and a decrease in PARP cleavage (82-kDa cleaved PARP; Fig. 6A, third panel, and Fig. 7D) in p53 siRNA-transfected cells compared with nontargeting siRNA-transfected cells. However, cleavage of PARP was not completely inhibited in the presence of p53 siRNA suggesting that apoptotic degradation of PARP is partially dependent on p53. These data indicate that p53 is required for daunomycin-induced apoptosis in MCF-7 cells. Therefore, the data generated using Trx overexpression suggest that the apoptotic potential of MCF-7 could be increased in the presence of higher levels of redox-active Trx. Because daunomycin is a quinone-containing anthracycline and undergoes redox cycling that generates ROS and the semiquinone radical, we tested whether Trx could contribute to enhanced redox cycling in vivo that could increase the expression of p53-mediated apoptosis.
Daunomycin Treatment Increases the Generation of ROS in Trx9 Cells-We estimated the total load of oxidative stress by using the ROSsensitive probe DCF-DA to measure the total cellular peroxide levels in vector, Trx9, or Serb4 cells in response to daunomycin. Preincubation of cells with non-fluorescent DCF-DA dye followed by treatment with daunomycin resulted in enhanced fluorescence due to increased oxidation of DCF-DA in Trx9 cells compared with vector or Serb4 cells (Fig.  8, A and B). We also used 5-iminodaunomycin, a non-redox cycling form of daunomycin, as a negative control (Fig. 8, A and C). 5-Iminodaunomycin does not contain the quinone moiety of anthracyclines and therefore does not generate superoxide anions (42). Treatment with 5-iminodaunomycin did not induce any detectable oxidation of DCF-DA in any of these Trx clones (Fig. 8, A and C). These data demonstrate that daunomycin could undergo a higher rate of redox cycling in the presence of a higher level of Trx.
Redox cycling of daunomycin generates semiquinone radical and O 2 . , both of which could induce DNA damage and cell death. Because Trx9 cells showed enhanced DNA damage and apoptosis, we sought to determine the rate of redox cycling of daunomycin in Trx9 cells. We measured the production of O 2 . in the presence of daunomycin in Trx clones as an indicator of redox cycling (43). As shown in Fig. 8D, treatment of cells with daunomycin produced a significantly higher level of O 2 . in Trx9 cells compared with vector or Serb4 cells. We have shown previously that Trx is an antioxidant, and it can scavenge hydroxyl radicals; however, it does not scavenge O 2 . (4). In addition, we also have shown that Escherichia coli Trx could participate in the redox cycling of anthracyclines generating O 2 . (44). Therefore, we further investigated whether human Trx could also cause redox cycling of daunomycin using additional controls. As demonstrated in Fig. 8D, treatment of MCF-7 clones with 5-iminodaunomycin did not increase the generation of superoxide anions in Trx9 or Serb4 cells. These data demonstrate that quinone redox cycling is enhanced in the presence of an increased level of Trx. Next we evaluated the effect of transient overexpression of Trx on daunomycin redox cycling. As demonstrated in Fig.  8E, MCF-7 cells expressing a higher level of Trx by adenovirus-mediated overexpression generated a higher level of superoxide anions compared with vector only-or dnTrx-transfected cells. In additional experiments (Fig. 8F) we down-regulated Trx expression using the siRNA  DECEMBER 2, 2005 • VOLUME 280 • NUMBER 48 approach and evaluated the response of these cells to generate superoxide anion in the presence of daunomycin. MCF-7 cells treated with siRNA demonstrated a lower level of superoxide anion generation in response to daunomycin. These data demonstrate that endogenous Trx is required for the redox cycling of daunomycin, and overexpression of Trx could enhance the level of redox cycling in these cells.

Thioredoxin Enhances Apoptosis
To determine whether the response obtained in MCF-7 cells with respect to generation of superoxide anions could be reproduced in other cell types, we down-regulated Trx expression in the lung adenocarcinoma cell line A549 using the siRNA approach and evaluated the response of these cells to daunomycin-mediated superoxide anion generation. As demonstrated in Fig. 8G, there was significant inhibition of superoxide anion generation in cell with a reduced level of Trx. These data indicate that endogenous Trx is required in cancer cells for redox cycling of anthracyclines.

Effect of Bioreductive Enzyme Inhibitor on O 2 . Anion Generation in
Trx9 Cells-We observed that Trx9 cells generate more O 2 . in the presence of anthracyclines. Redox cycling of anthracyclines has been shown to be mediated by one electron reduction by NADPH-cytochrome P-450 reductase (45), mitochondrial NADH dehydrogenase (46), and nitrate reductase (47) from Neurospora. Therefore, to determine the involvement of bioreductive enzyme(s) in the redox cycling of daunomycin in the presence of increased thioredoxin we treated cells with diphenyleneiodonium chloride (DPIC), a nonspecific inhibitor of NADPH-dependent reductases (48). As demonstrated in Fig. 9, treatment of cells with DPIC inhibited the generation of O 2 . suggesting that Trx-mediated O 2 . generation is dependent on bioreductive enzymes for anthracycline redox cycling. These results indicate that reductases could be involved in the redox cycling of anthracyclines using reducing equivalents from reduced Trx. Redox Cycling Contributes to Increased p53-DNA Binding in the Presence of a Higher Level of Redox-active Trx-Next we determined whether the increase in p53-DNA binding observed in Trx9 cells depends on the redox cycling of daunomycin. By treating Trx clones with 5-iminodaunomycin, which cannot undergo redox cycling but intercalates with DNA, we show that each of these clones (vector, Trx9, and Serb4) exhibited enhanced p53-DNA binding (Fig. 10A). However, there were minor differences in the intensity of DNA binding in these cells. To determine whether ROS mediate p53-DNA binding due to daunomycin redox cycling we used H 2 O 2 as a positive control for ROS and Taxol as a negative control for ROS. Additionally we used menadione as a positive control for quinone-containing compound. As shown in Fig. 10B, H 2 O 2 , menadione, and Taxol treatment did not induce p53-DNA binding in Trx clones except that 500 M H 2 O 2 showed very weak p53-DNA binding (Fig. 10B). These data indicate that either H 2 O 2 is scavenged by peroxiredoxins that have been shown to be up-regulated in Trx9 cells (6) or that H 2 O 2 is not an effective inducer of p53 response. However, menadione, which contains a quinone moiety, also did not show p53-DNA binding in our protocol, suggesting that daunomycin or doxorubicin could specifically interact with Trx. To further evaluate the role of ROS in p53-DNA binding, we treated Trx9 cells with other ROS-generating compounds such as pyocyanin or phenazine methosulfate (49,50). We did not observe any detectable increase in p53-DNA binding (Fig. 10C). These studies indicate that ROS may not contribute substantially to p53 activation in MCF-7 cells.
To understand the relative contribution of ROS or intercalation of drug due to redox cycling on p53 activation, we used MnTBAP to scavenge O 2 . or H 2 O 2 (51) in daunomycin-treated Trx9 cells. As demonstrated in Fig. 10C (upper panel), MnTBAP treatment resulted in about 30% (densitometric analysis) reduction in the p53 binding activity.
These data indicate that activation of p53 is not fully dependent on ROS. Because Ser 15 phosphorylation on p53 enables it as a DNA binding transcription factor we also determined Ser 15 phosphorylation in response to MnTBAP treatment. As demonstrated in Fig. 10C (lower  panel, lanes 3-4), there was no appreciable decrease in Ser 15 phosphorylation in response to MnTBAP in daunomycin-treated cells. These data further support the notion that ROS is not a major inducer of p53 activation due to redox cycling of daunomycin but rather due to DNA damage by the interaction of the drug with DNA. Next we determined the role of reductases in redox cycling of anthracyclines. Treatment of cells with the general reductase inhibitor DPIC abolished p53-DNA binding as well as p53 (Ser 15 ) phosphorylation (Fig. 10C, lanes 5-6). These data indicate that generation of semiquinone by redox cycling may contribute to p53 activation as a result of DNA damage in Trx9 cells and that ROS plays a minor role in the activation of p53.

DISCUSSION
In the present study we demonstrated that increased expression of Trx enhances apoptosis in MCF-7 cells in response to daunomycin. In contrast, silencing endogenous Trx expression resulted in a significant decrease in p53 expression and apoptosis in MCF-7 cells as well as other cells in response to daunomycin. The expression and DNA binding of p53 protein was also increased in response to daunomycin in Trx9 cells compared with vector or Serb4 cells. Additionally there was increased production of O 2 . in extracts of Trx9 cells in response to daunomycin.
DPIC, a general reductase inhibitor, decreased the generation of O 2 . as well as p53-DNA binding in Trx9 cells in response to daunomycin. Taken together, our studies demonstrate that Trx increases the redox cycling of daunomycin and enhances the apoptotic potential of daunomycin. Our data also indicate that endogenous Trx is essential for anthracycline-dependent p53 expression and the activation of the apoptotic cascade. These are novel pro-oxidant and proapoptotic roles of Trx in response to anthracycline drugs. Additionally the present study also indicates a crucial role of Trx in daunomycin-induced caspase-1 expression.
Thioredoxin is widely considered to be an antioxidant protecting cells from a variety of oxidative stress conditions (1,2). Our data indicate that Trx could provide reducing equivalents to bioreductive enzymes that play a role in redox cycling of anthracyclines, and this process increases the apoptotic potential of anthracyclines. Interestingly Trx has been shown to confer resistance against ROS-generating anticancer drugs because of its antioxidant properties (31,33). Conversely recent studies also have shown that Trx does not confer resistance against doxorubicin (6). Furthermore thioredoxin failed to protect MCF-7 cells from apoptosis induced by ROS-generating drugs. The study published by Wang et al. (52) is a correlative study that demonstrated a correlation between increased Trx expression in various leukemia cell lines and the drug resistance to adriamycin. Therefore, there was no mechanistic evaluation of the role of Trx in drug resistance or apoptosis. The study also compared several cell lines in terms of Trx expression and drug resistance. Our present investigation showed, using different expression systems and enhancing endogenous Trx in multiple cells, that increased Trx expression could enhance the apoptotic potential of anthracyclines. Additionally the role of endogenous Trx in apoptosis of cancer cells in response to daunomycin was clearly elucidated using an siRNA approach. In contrast to the study of Wang et al. (52), studies by Berggren et al. (6) have demonstrated clearly that although Trx protects cells against H 2 O 2 -mediated apoptosis, it does not protect against adriamycin-induced apoptosis. They also demonstrated that the protective effect of Trx against H 2 O 2 is due to enhanced peroxiredoxin expression (6). This study clearly demonstrated that Trx could remove H 2 O 2 due to higher peroxiredoxin activity, but it cannot protect against doxorubicin-induced toxicity (6). These results support our data that H 2 O 2 does not play a major role in apoptosis of daunomycin-treated, Trx-overexpressing cells. Therefore, if H 2 O 2 were the only cytotoxic compound that mediates doxorubicin-induced apoptosis then probably Trx overexpression would protect against doxorubicin-induced apoptosis. However, the toxicity of anthracyclines predominantly comes from the semiquinone intercalation with the DNA resulting in DNA damage and apoptosis. In our studies we observed that Trx9 cells not only failed to protect against daunomycin-mediated apoptosis but also enhanced apoptosis in these cells. Thus, data obtained using multiple systems using different approaches confirm a crucial role of Trx in the apoptotic response of cancer cells in treatment with daunomycin. Taken together, these data indicate that a protective role of Trx could be important in ROS-mediated cytotoxicity. As demonstrated in our data, removal of superoxide anion or H 2 O 2 by using MnTBAP decreased p53-DNA binding by about 25-30%. Additionally treating cells with H 2 O 2 or other ROS-generating systems did not induce significant p53-DNA binding. This fact demonstrates that ROS are generated due to redox cycling, but they do not significantly contribute to the p53-dependent apoptotic process. Therefore, Trx could inhibit the ROS-mediated cytotoxicity such as that of H 2 O 2 as has been shown, but it does not inhibit the redox cycling process that facilitates the intercalation of the drug with the DNA.
We have shown earlier that Trx induces manganese superoxide dismutase (5). Additionally Trx does not scavenge O 2 . (4). However, it can scavenge hydroxyl radicals or singlet oxygen (4). O 2 . is the first reduction product of redox cycling that could produce H 2 O 2 , which could be scavenged by peroxiredoxin or other peroxidases. Therefore, in this circumstance the redox cycling-enhancing action of Trx by its electrondonating function will remain unaffected. Additionally our data indicated that NADPH-dependent reductase inhibitor inhibited O 2 . generation in Trx9 cells, suggesting that redox cycling produces enhanced DNA damage in Trx9 cells resulting in p53 activation. These findings are further verified by the fact that dominant negative expression of redox-inactive mutant Trx or silencing of Trx using Trx siRNA failed to induce p53 protein in response to daunomycin suggesting that Trx is required for bioreductive activation of daunomycin. Our data presented here indicate a fundamental role of endogenous Trx in redox cycling of anthracyclines, which has not been recognized previously. We have shown previously that E. coli Trx could enhance redox cycling of anthracyclines and induce DNA damage both in vivo and in vitro (44). We have also shown that E. coli Trx provides reducing equivalents to Trx reductase and mammalian cytochrome P-450 reductase that enhanced the redox cycling of anthracyclines (44). It is therefore conceivable that Trx may indirectly enhance redox cycling of daunomycin involving NADPH-dependent reductase. We also demonstrated that DPIC abolished daunomycin-induced p53-DNA binding, suggesting that redox cycling of daunomycin depends on NADPH-dependent bioreductive enzymes.
Although daunomycin potently induced p53-DNA binding, other ROS-generating agents including menadione did not induce p53-DNA binding (Fig. 10B) at the concentrations used in our study. A previous study also observed failure of menadione to induce p53-DNA binding in MCF-7 cells (18). However, p53 expression did increase several hours after menadione was removed, indicating that p53 was activated in the DNA damage repair phase (18). Thus, it is unclear why menadione did not activate p53, whereas daunomycin did induce p53 activation. H 2 O 2 did not induce a strong p53 response at 500 M concentration. Increased Trx expression could have increased H 2 O 2 scavenging by peroxiredoxins, which obtain reducing equivalents from Trx. A lower level of p53-DNA binding was observed in Trx mutant Serb4 cells that express redox-inactive form of Trx, which does not show higher peroxiredoxin expression (6). This fact suggests that removal of H 2 O 2 by overexpression of redox-active Trx in Trx9 cells could not have accounted for less p53-DNA binding in response to H 2 O 2 . Furthermore DNA intercalation appears to be essential for induction of p53-DNA binding because 5-iminodaunomycin did induce p53-DNA binding. This fact was further strengthened because there was no involvement of redox cycling in 5-iminodaunomycin, and p53-DNA binding was unaffected in both Trx9 and Serb4 cells.
We also demonstrated decreased O 2 . production, p53 expression, and apoptosis in daunomycin-treated MCF-7 cells deficient in Trx using Trx siRNA. Accumulating evidence from various studies indicates that p53 is required to enhance chemosensitivity in cancer cells (38,39). In the present study we used MCF-7 cells, which are defective in caspase-3 and do not undergo classical 180-bp DNA fragmentation during apoptosis (13) and therefore are dependent on alternate or compensatory mechanisms to induce apoptosis. Furthermore in p53-defective cells there are alternate regulatory proteins such as Rb or E2F that could trigger apoptosis. In the present context it becomes more important for cells such as the caspase-3-deficient MCF-7 cell line with wild type p53 to utilize a p53-mediated response to induce apoptosis. In summary, our investigation demonstrates a novel pro-oxidant and proapoptotic role of Trx in response to the anthracycline class of antitumor agents.