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• W1987945844 abstract "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 (O2˙-) in anthracycline-treated cell extracts. Enhanced generation of O2˙- 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. 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 (O2˙-) in anthracycline-treated cell extracts. Enhanced generation of O2˙- 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) 2The abbreviations used are: TrxthioredoxindnTrxdominant negative TrxsiRNAsmall interfering RNAPARPpoly(ADP-ribose) polymeraseROSreactive oxygen speciesTUNELterminal deoxynucleotidyl transferase dUTP nick-end labelingPBSphosphate-buffered salineDCF-DA2′,7′-dichlorofluorescin diacetateEMSAelectrophoretic mobility shift assayDPICdiphenyleneiodonium chlorideMnTBAPmanganese(III) meso-tetrakis(4-benzoic acid)porphyrin. 2The abbreviations used are: TrxthioredoxindnTrxdominant negative TrxsiRNAsmall interfering RNAPARPpoly(ADP-ribose) polymeraseROSreactive oxygen speciesTUNELterminal deoxynucleotidyl transferase dUTP nick-end labelingPBSphosphate-buffered salineDCF-DA2′,7′-dichlorofluorescin diacetateEMSAelectrophoretic mobility shift assayDPICdiphenyleneiodonium chlorideMnTBAPmanganese(III) meso-tetrakis(4-benzoic acid)porphyrin. is a low molecular mass protein (12 kDa) that is widely distributed; Trx is found within the cytoplasmic, membrane, extracellular, and mitochondrial cellular fractions (1Holmgren A. Bjornstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (809) Google Scholar, 2Powis G. Montfort W.R. Annu. Rev. Biophys. Biomol. Struct. 2001; 30: 421-455Crossref PubMed Scopus (274) Google Scholar). 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 (3Mitsui A. Hirakawa T. Yodoi J. Biochem. Biophys. Res. Commun. 1992; 186: 1220-1226Crossref PubMed Scopus (172) Google Scholar, 4Das K.C. Das C.K. Biochem. Biophys. Res. Commun. 2000; 277: 443-447Crossref PubMed Scopus (203) Google Scholar), Trx also plays an important role in regulating the expression of other antioxidant genes such as manganese superoxide dismutase (5Das K.C. Lewis-Molock Y. White C.W. Am. J. Respir. Cell Mol. Biol. 1997; 17: 713-726Crossref PubMed Scopus (115) Google Scholar). Trx overexpression also enhances the expression of peroxiredoxin that could reduce peroxides to molecular oxygen and H2O (6Berggren M.I. Husbeck B. Samulitis B. Baker A.F. Gallegos A. Powis G. Arch. Biochem. Biophys. 2001; 392: 103-109Crossref PubMed Scopus (102) Google Scholar). Trx has been shown to regenerate oxidatively inactivated proteins (7Fernando M.R. Nanri H. Yoshitake S. Nagata-Kuno K. Minakami S. Eur. J. Biochem. 1992; 209: 917-922Crossref PubMed Scopus (200) Google Scholar, 8Spector A. Yan G.Z. Huang R.R. McDermott M.J. Gascoyne P.R. Pigiet V. J. Biol. Chem. 1988; 263: 4984-4990Abstract Full Text PDF PubMed Google Scholar). In addition to its role as an antioxidant protein, Trx has been shown to have growth promoting properties (9Powis G. Mustacich D. Coon A. Free Radic. Biol. Med. 2000; 29: 312-322Crossref PubMed Scopus (365) Google Scholar). In contrast, a recent study has demonstrated that overexpression of redox-active Trx could promote cell death via activation of caspase-8 (10Ma X. Karra S. Lindner D.J. Hu J. Reddy S.P. Kimchi A. Yodoi J. Kalvakolanu D.V. Oncogene. 2001; 20: 3703-3715Crossref PubMed Scopus (18) Google Scholar). Additional studies have shown that Trx reductase is critical for cell death, and a Trx-dependent mechanism has been suggested (11Ueda S. Masutani H. Nakamura H. Tanaka T. Ueno M. Yodoi J. Antioxid. Redox Signal. 2002; 4: 405-414Crossref PubMed Scopus (459) Google Scholar). Recent studies also indicate that caspases, the executioner of cell death by apoptosis, could be activated by Trx due to its disulfide reducing properties (12Ueda S. Nakamura H. Masutani H. Sasada T. Yonehara S. Takabayashi A. Yamaoka Y. Yodoi J. J. Immunol. 1998; 161: 6689-6695PubMed Google Scholar). 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 (12Ueda S. Nakamura H. Masutani H. Sasada T. Yonehara S. Takabayashi A. Yamaoka Y. Yodoi J. J. Immunol. 1998; 161: 6689-6695PubMed Google Scholar). Furthermore Trx also has been shown to promote p53-DNA binding due to its reducing actions on DNA-binding cysteine motifs on p53 (14Ueno M. Masutani H. Arai R.J. Yamauchi A. Hirota K. Sakai T. Inamoto T. Yamaoka Y. Yodoi J. Nikaido T. J. Biol. Chem. 1999; 274: 35809-35815Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). 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 (5Das K.C. Lewis-Molock Y. White C.W. Am. J. Respir. Cell Mol. Biol. 1997; 17: 713-726Crossref PubMed Scopus (115) Google Scholar, 15Andoh T. Chock P.B. Chiueh C.C. J. Biol. Chem. 2002; 277: 9655-9660Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) as well as direct scavenging of hydroxyl radicals or singlet oxygen. thioredoxin dominant negative Trx small interfering RNA poly(ADP-ribose) polymerase reactive oxygen species terminal deoxynucleotidyl transferase dUTP nick-end labeling phosphate-buffered saline 2′,7′-dichlorofluorescin diacetate electrophoretic mobility shift assay diphenyleneiodonium chloride manganese(III) meso-tetrakis(4-benzoic acid)porphyrin. thioredoxin dominant negative Trx small interfering RNA poly(ADP-ribose) polymerase reactive oxygen species terminal deoxynucleotidyl transferase dUTP nick-end labeling phosphate-buffered saline 2′,7′-dichlorofluorescin diacetate electrophoretic mobility shift assay diphenyleneiodonium chloride manganese(III) meso-tetrakis(4-benzoic acid)porphyrin. The anthracycline class of anticancer drugs such as doxorubicin or daunomycin has been shown to induce p53-dependent apoptosis in cancer cells (16Cho Y. Hokkaido Igaku Zasshi. 1999; 74: 239-248PubMed Google Scholar, 17Asher G. Lotem J. Cohen B. Sachs L. Shaul Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1188-1193Crossref PubMed Scopus (288) Google Scholar). Additionally anthracyclines have also been shown to cause DNA damage, which increases p53 expression (18Ngo E.O. Nutter L.M. Sura T. Gutierrez P.L. Chem. Res. Toxicol. 1998; 11: 360-368Crossref PubMed Scopus (20) Google Scholar, 19Sadji-Ouatas Z. Lasfer M. Julien S. Feldmann G. Reyl-Desmars F. Biochem. J. 2002; 364: 881-885Crossref PubMed Scopus (19) Google Scholar). 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 (20Maya R. Balass M. Kim S.T. Shkedy D. Leal J.F. Shifman O. Moas M. Buschmann T. Ronai Z. Shiloh Y. Kastan M.B. Katzir E. Oren M. Genes Dev. 2001; 15: 1067-1077Crossref PubMed Scopus (523) Google Scholar). Phosphorylation of p53 on the Ser15 residue dissociates MDM2 and activates p53 as a transcription factor, which binds to various p53-dependent genes resulting in their activation or repression (20Maya R. Balass M. Kim S.T. Shkedy D. Leal J.F. Shifman O. Moas M. Buschmann T. Ronai Z. Shiloh Y. Kastan M.B. Katzir E. Oren M. Genes Dev. 2001; 15: 1067-1077Crossref PubMed Scopus (523) Google Scholar). 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 (21Iyanagi T. Yamazaki I. Biochim. Biophys. Acta. 1969; 172: 370-381Crossref PubMed Scopus (134) Google Scholar, 22Bolton J.L. Trush M.A. Penning T.M. Dryhurst G. Monks T.J. Chem. Res. Toxicol. 2000; 13: 135-160Crossref PubMed Scopus (1354) Google Scholar, 23Iyanagi T. Yamazaki I. Biochim. Biophys. Acta. 1970; 216: 282-294Crossref PubMed Scopus (306) Google Scholar). This bioreductive process generates semiquinone radical with concomitant production of superoxide anion (O2˙-). The semiquinone radical intercalates with the DNA resulting in DNA damage. The formation of O2˙- is the beginning of a cascade that generates hydrogen peroxide and hydroxyl radicals, generally referred to as reactive oxygen species (24Mordente A. Meucci E. Martorana G.E. Giardina B. Minotti G. IUBMB Life. 2001; 52: 83-88Crossref PubMed Scopus (71) Google Scholar). In addition to various bioreductive enzymes, low molecular weight protein or non-protein thiols may also take part in the redox cycling process (25Hrdina R. Gersl V. Klimtova I. Simunek T. Machackova J. Adamcova M. Acta Med. (Hradec Kralove). 2000; 43: 75-82Crossref PubMed Google Scholar). 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. Reagents—Daunomycin was purchased from Sigma, and 5-iminodaunomycin was obtained from NCI, National Institutes of Health. Anti-p53 (full length), anti-caspase-7, and anti-caspase-1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); anti-p53 phospho-Ser15, anti-caspase-6, anti-caspase-8 (recognizes cleaved fragment), and anti-poly(ADP-ribose) polymerase (PARP) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Antithioredoxin antibody was purchased from Research Diagnostics (Flanders, NJ). 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 (26Oblong J.E. Berggren M. Gasdaska P.Y. Powis G. J. Biol. Chem. 1994; 269: 11714-11720Abstract Full Text PDF PubMed Google Scholar, 27Gallegos A. Gasdaska J.R. Taylor C.W. Paine-Murrieta G.D. Goodman D. Gasdaska P.Y. Berggren M. Briehl M.M. Powis G. Cancer Res. 1996; 56: 5765-5770PubMed Google Scholar). MCF-7 clones were cultured in Dulbecco's modified Eagle's medium containing G418 (300 μg/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 (26Oblong J.E. Berggren M. Gasdaska P.Y. Powis G. J. Biol. Chem. 1994; 269: 11714-11720Abstract Full Text PDF PubMed Google Scholar) 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 ∼1 × 108 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-mm2 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 (1Holmgren A. Bjornstedt M. Methods Enzymol. 1995; 252: 199-208Crossref PubMed Scopus (809) Google Scholar). Briefly the reaction mixture contained NADPH (200 μm), porcine insulin (80 μm, Sigma), and bovine Trx reductase (0.1 μm) 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 μm). 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 3′-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 × 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 -20 °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 G1” peak (6Berggren M.I. Husbeck B. Samulitis B. Baker A.F. Gallegos A. Powis G. Arch. Biochem. Biophys. 2001; 392: 103-109Crossref PubMed Scopus (102) Google Scholar) 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 of O2˙- Production by Reduction of Ferricytochrome c—Superoxide production was measured as superoxide dismutase-inhibitable reduction of ferricytochrome c (28Azzi A. Montecucco C. Richter C. Biochem. Biophys. Res. Commun. 1975; 65: 597-603Crossref PubMed Scopus (299) Google Scholar). Cells were sonicated in potassium phosphate buffer (0.05 m, pH 7.8, plus 1 mm EDTA) and centrifuged, and the supernatant was used for the assay. To determine the O2˙- generation in the cell lysate, the supernatant was incubated with 10 μm drug and 10 μm cytochrome c with or without 1 unit of superoxide dismutase to determine superoxide dismutase inhibitable rate. All reactions were performed in triplicate. The reduction of ferricytochrome c was measured both in kinetic and end point mode with path check on for 1-h duration at a wavelength 550 nm using a Spectramax 190 plate reader (Molecular Devices). Total protein was quantified using the Bradford protein assay (Bio-Rad). In Situ Detection of O2˙- by Fluorescent Probe 2′,7′-Dichlorofluorescin Diacetate (DCF-DA)—Cells were grown in chambered glass slides (Nunc) to a final density of 25,000 cells/well. Cells were preincubated with 20 μm DCF-DA (Sigma) in 20 mm HEPES in PBS containing 5 mg/ml bovine serum albumin at 37 °C for 30 min followed by washing with PBS buffer, the drug was added, and cells were observed for 300 s in a Nikon laser confocal microscope using a laser beam wavelength of 488 nm and analyzed by Ultraview software (PerkinElmer Life Sciences). Clonogenic Assay—Cells were trypsinized and seeded to a final density of 1 × 106 viable cells/100-mm2 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-mm2 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 (29Das K.C. J. Biol. Chem. 2001; 276: 4662-4670Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Briefly cells were washed in ice-cold PBS and harvested in 2 ml of ice-cold PBS by centrifugation. Cell pellets were resuspended in 400 μl 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 μg/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 -70 °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 (5′-GGCATGTCCGGGCATGTCC-3′) and was end-labeled using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [γ-32P]ATP (PerkinElmer Life Sciences) in 10× kinase buffer supplied with the enzyme. Ten micrograms of nuclear protein were preincubated in 5 μl of 5× binding buffer (20% glycerol, 5 mm MgCl2, 5 mm EDTA, 5 mm dithiothreitol, 500 mm NaCl, 50 mm Tris·HCl, 0.4 mg/ml calf thymus DNA), 200 ng of anti-p53 polyclonal antibody 421 and 2 μg 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 0.25× 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 Tris·HCl (pH 7.5) containing 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, and 20 μg/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 (30Das K.C. White C.W. J. Immunol. Methods. 1998; 211: 9-20Crossref PubMed Scopus (17) Google Scholar). 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 (31Sasada T. Iwata S. Sato N. Kitaoka Y. Hirota K. Nakamura K. Nishiyama A. Taniguchi Y. Takabayashi A. Yodoi J. J. Clin. Investig. 1996; 97: 2268-2276Crossref PubMed Scopus (182) Google Scholar, 32Sasada T. Nakamura H. Ueda S. Sato N. Kitaoka Y. Gon Y. Takabayashi A. Spyrou G. Holmgren A. Yodoi J. Free Radic. Biol. Med. 1999; 27: 504-514Crossref PubMed Scopus (108) Google Scholar, 33Yokomizo A. Ono M. Nanri H. Makino Y. Ohga T. Wada M. Okamoto T. Yodoi J. Kuwano M. Kohno K. Cancer Res. 1995; 55: 4293-4296PubMed Google Scholar). 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 classical DNA laddering during apoptosis. Therefore, we further determined apoptotic cells as the “sub-G1” 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 (34Islam M.M. Begum S. Lin L. Okamura A. Du M. Fujimura S. Cancer Chemother. Pharmacol. 2002; 49: 111-118Crossref PubMed Scopus (9) Google Scholar, 35Munshi A. Hobbs M. Meyn R.E. Methods Mol. Med. 2005; 110: 21-28PubMed Google Scholar). Therefore we compared the clonogenic survival of Trx9, Serb4, or vector cells treated with daunomycin at concentrations as low as 0.025 μm. A clonogenic assay is a more stringent assessment of chemosensitivity than TUNEL or sub-G1 peak measurements (34Islam M.M. Begum S. Lin L. Okamura A. Du M. Fujimura S. Cancer Chemother. Pharmacol. 2002; 49: 111-118Crossref PubMed Scopus (9) Google Scholar, 35Munshi A. Hobbs M. Meyn R.E. Methods Mol. Med. 2005; 110: 21-28PubMed Google Scholar). Trx9 cells treated with daunomycin formed significantly less colonies at the end of 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 dnTrx-overexpressing 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 non-targeting 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 Fig. 3C, MCF-7 cells transfected with non-targeting siRNA and treated with daunomycin demonstrated a significant increase in p53 protein level. In contrast, cells treated with Trx siRNA demonstrated a significantly (p < 0.001) lower level of p53 protein in response to daunomycin treatment (Fig. 3C). Additionally the level of active caspase-7 (Fig. 3B, second panel, and Fig. 3D) and the level of cleaved PARP (Fig. 3B,third panel, and Fig. 3E) were also significantly (p < 0.001) decreased in Trx siRNA-transfected cells compared with non-targeting siRNA-transfected cells. These data demonstrate a crucial role of endogenous Trx in daunomycin-induced apoptosis in MCF-7 cells. Recent studies have demonstrated that caspase-1 expression is upregulated in response to doxorubicin in a p53-dependent manner in MCF-7 cells (36Gupta S. Radha V. Furukawa Y. Swarup G. J. Biol. Chem. 2001; 276: 10585-10588Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 37Sadasivam S. Gupta S. Radha V. Batta K. Kundu T.K. Swarup G. Oncogene. 2005; 24: 627-636Crossref PubMed Scopus (44) Google Scholar). 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 re" @default.
• W1987945844 created "2016-06-24" @default.
• W1987945844 creator A5065370839 @default.
• W1987945844 creator A5078559032 @default.
• W1987945844 creator A5089042215 @default.
• W1987945844 date "2005-12-01" @default.
• W1987945844 modified "2023-10-17" @default.
• W1987945844 title "Endogenous Thioredoxin Is Required for Redox Cycling of Anthracyclines and p53-dependent Apoptosis in Cancer Cells" @default.
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