Dual targeting of the thioredoxin and glutathione antioxidant systems in malignant B cells: A novel synergistic therapeutic approach
2014; Elsevier BV; Volume: 43; Issue: 2 Linguagem: Inglês
10.1016/j.exphem.2014.10.004
ISSN1873-2399
AutoresMichelle Kiebala, Jolanta Skalska, Carla Casulo, Paul S. Brookes, Derick R. Peterson, Shannon P. Hilchey, Yun Dai, Steven Grant, Sanjay B. Maggirwar, Steven H. Bernstein,
Tópico(s)Sulfur Compounds in Biology
Resumo•B-cell malignancies are a common type of cancer.•We investigated the effect of auranofin + BSO on malignant B-cell survival.•AUR interacted synergistically with BSO to trigger death in malignant B-cell types.•There was less toxicity toward normal B-cells.•Toxicity is mediated by dual inhibition of TrxR and NF-κB. B-cell malignancies are a common type of cancer. One approach to cancer therapy is to either increase oxidative stress or inhibit the stress response systems on which cancer cells rely. In this study, we combined nontoxic concentrations of Auranofin (AUR), an inhibitor of the thioredoxin system, with nontoxic concentrations of buthionine-sulfoximine (BSO), a compound that reduces intracellular glutathione levels, and investigated the effect of this drug combination on multiple pathways critical for malignant B-cell survival. Auranofin interacted synergistically with BSO at low concentrations to trigger death in multiple malignant B-cell lines and primary mantle-cell lymphoma cells. Additionally, there was less toxicity toward normal B cells. Low AUR concentrations inhibited thioredoxin reductase (TrxR) activity, an effect significantly increased by BSO cotreatment. Overexpression of TrxR partially reversed AUR+BSO toxicity. Interestingly, the combination of AUR+BSO inhibited nuclear factor κB (NF-κB) signaling. Moreover, synergistic cell death induced by this regimen was attenuated in cells overexpressing NF-κB proteins, arguing for a functional role for NF-κB inhibition in AUR+BSO-mediated cell death. Together, these findings suggest that AUR+BSO synergistically induces malignant B-cell death, a process mediated by dual inhibition of TrxR and NF-κB, and such an approach warrants further investigation in B-cell malignancies. B-cell malignancies are a common type of cancer. One approach to cancer therapy is to either increase oxidative stress or inhibit the stress response systems on which cancer cells rely. In this study, we combined nontoxic concentrations of Auranofin (AUR), an inhibitor of the thioredoxin system, with nontoxic concentrations of buthionine-sulfoximine (BSO), a compound that reduces intracellular glutathione levels, and investigated the effect of this drug combination on multiple pathways critical for malignant B-cell survival. Auranofin interacted synergistically with BSO at low concentrations to trigger death in multiple malignant B-cell lines and primary mantle-cell lymphoma cells. Additionally, there was less toxicity toward normal B cells. Low AUR concentrations inhibited thioredoxin reductase (TrxR) activity, an effect significantly increased by BSO cotreatment. Overexpression of TrxR partially reversed AUR+BSO toxicity. Interestingly, the combination of AUR+BSO inhibited nuclear factor κB (NF-κB) signaling. Moreover, synergistic cell death induced by this regimen was attenuated in cells overexpressing NF-κB proteins, arguing for a functional role for NF-κB inhibition in AUR+BSO-mediated cell death. Together, these findings suggest that AUR+BSO synergistically induces malignant B-cell death, a process mediated by dual inhibition of TrxR and NF-κB, and such an approach warrants further investigation in B-cell malignancies. Although the survival of patients suffering from B-cell lymphoproliferative disorders has improved in recent years, many ultimately succumb to their diseases. Consequently, newer and more effective therapeutic approaches are urgently needed. One therapeutic approach is based on the concept that neoplastic cells require a higher level of oxidative stress than their normal cellular counterparts to maintain their neoplastic phenotype. Such cells have been described as being nononcogene addicted to oxidative stress [1Sharma S.V. Settleman J. Exploiting the balance between life and death: Targeted cancer therapy and "oncogenic shock".Biochem Pharmacol. 2010; 80: 666-673Crossref PubMed Scopus (47) Google Scholar, 2Luo J. Solimini N.L. Elledge S.J. Principles of cancer therapy: Oncogene and nononcogene addiction.Cell. 2009; 136: 823-837Abstract Full Text Full Text PDF PubMed Scopus (1339) Google Scholar]. To survive greater degrees of oxidative stress, cancer cells are more dependent than their normal counterparts on stress response systems, such as the thioredoxin (Trx) and glutathione (GSH) systems. Therefore, one approach to cancer therapy involves targeting this oxidative stress addiction by either increasing oxidative stress or inhibiting the stress response systems [1Sharma S.V. Settleman J. Exploiting the balance between life and death: Targeted cancer therapy and "oncogenic shock".Biochem Pharmacol. 2010; 80: 666-673Crossref PubMed Scopus (47) Google Scholar]. Given that the neoplastic cells are closer to the oxidative stress lethal threshold than their normal counterparts, this approach has the potential for cancer-selective lethality.Auranofin (AUR), a sulfur-containing, oral, gold(I)-based agent, has an excellent safety profile and was approved by the United States Food and Drug Administration in 1985 for the treatment of patients with rheumatoid arthritis [3Champion G.D. Cairns D.R. Bieri D. et al.Dose response studies and longterm evaluation of auranofin in rheumatoid arthritis.J Rheumatol. 1988; 15: 28-34PubMed Google Scholar, 4Williams H.J. Ward J.R. Egger M.J. et al.Auranofin, gold sodium thiomalate, and placebo in the treatment of rheumatoid arthritis. Cooperative systematic studies of rheumatic diseases.Clin Rheumatol. 1984; 3: 39-50Crossref PubMed Scopus (5) Google Scholar, 5Furst D.E. Mechanism of action, pharmacology, clinical efficacy and side effects of auranofin. An orally administered organic gold compound for the treatment of rheumatoid arthritis.Pharmacotherapy. 1983; 3: 284-298PubMed Google Scholar, 6Brewer Jr., E.J. Giannini E.H. Person D.A. Early experiences with auranofin in juvenile rheumatoid arthritis.Am J Med. 1983; 75: 152-156Abstract Full Text PDF PubMed Scopus (18) Google Scholar, 7Hull R.G. Morgan S.H. Parke A.L. Childs L. Goldman M. Hughes G.R. A double-blind study comparing sodium aurothiomalate and auranofin in patients with rheumatoid arthritis previously stabilized on sodium aurothiomalate.Int J Clin Pharmacol Res. 1984; 4: 395-401PubMed Google Scholar, 8Jessop J.D. O'Sullivan M.M. Lewis P.A. et al.A long-term five-year randomized controlled trial of hydroxychloroquine, sodium aurothiomalate, auranofin and penicillamine in the treatment of patients with rheumatoid arthritis.Br J Rheumatol. 1998; 37: 992-1002Crossref PubMed Scopus (42) Google Scholar]. Auranofin inhibits thioredoxin reductase (TrxR), a major cellular antioxidant protein that is a component of the cellular Trx system [9Mustacich D. Powis G. Thioredoxin reductase.Biochem J. 2000; : 1-8Crossref PubMed Scopus (752) Google Scholar]. Auranofin has been shown to induce apoptotic death of multiple human tumors including breast cancer [10Kim N.H. Park H.J. Oh M.K. Kim I.S. Antiproliferative effect of gold(I) compound auranofin through inhibition of STAT3 and telomerase activity in MDA-MB 231 human breast cancer cells.BMB Rep. 2013; 46: 59-64Crossref PubMed Scopus (50) Google Scholar], ovarian cancer [11Marzano C. Gandin V. Folda A. Scutari G. Bindoli A. Rigobello M.P. Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatin-resistant human ovarian cancer cells.Free Radic Biol Med. 2007; 42: 872-881Crossref PubMed Scopus (330) Google Scholar], multiple myeloma (MM) [12Nakaya A. Sagawa M. Muto A. Uchida H. Ikeda Y. Kizaki M. The gold compound auranofin induces apoptosis of human multiple myeloma cells through both down-regulation of STAT3 and inhibition of NF-kappaB activity.Leuk Res. 2011; 35: 243-249Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar], and chronic lymphocytic leukemia [13Zalewski P.D. Forbes I.J. Valente L. Hurst N.P. Auranofin increases the affinity of phorbol dibutyrate receptors in chronic lymphocytic leukemia cells (B cells).J Immunol. 1987; 138: 3005-3009PubMed Google Scholar].Homeostasis of the cellular redox state is regulated not only by the Trx-dependent system but also by the GSH system. Recent studies demonstrate that genetic ablation of TrxR1 results in a compensatory increase in the activity of the GSH system [14Mandal P.K. Schneider M. Kolle P. et al.Loss of thioredoxin reductase 1 renders tumors highly susceptible to pharmacologic glutathione deprivation.Cancer Res. 2010; 70: 9505-9514Crossref PubMed Scopus (100) Google Scholar], supporting the notion of redundancy in antioxidant pathways. Given this redundancy, it is not surprising that inhibition of the GSH system, in conjunction with genetic inhibition of either TrxR1 or TrxR2, results in the elicitation of pronounced neoplastic cell death [14Mandal P.K. Schneider M. Kolle P. et al.Loss of thioredoxin reductase 1 renders tumors highly susceptible to pharmacologic glutathione deprivation.Cancer Res. 2010; 70: 9505-9514Crossref PubMed Scopus (100) Google Scholar]. A pharmacologic approach to inhibit GSH is to use buthionine-sulfoximine (BSO), an inhibitor of the rate-limiting enzyme in GSH biosynthesis, γ-glutamylcysteine synthetase [15Griffith O.W. Mechanism of action, metabolism, and toxicity of buthionine sulfoximine and its higher homologs, potent inhibitors of glutathione synthesis.J Biol Chem. 1982; 257: 13704-13712Abstract Full Text PDF PubMed Google Scholar]. Therefore, combining AUR with agents that inhibit or deplete GSH is a rational therapeutic combination, and in fact, combinations of AUR and BSO have recently been shown to selectively kill human head, neck, and lung cancer cells by inducing oxidative stress [16Sobhakumari A. Love-Homan L. Fletcher E.V. et al.Susceptibility of human head and neck cancer cells to combined inhibition of glutathione and thioredoxin metabolism.PLoS One. 2012; 7: e48175Crossref PubMed Scopus (55) Google Scholar, 17Fath M.A. Ahmad I.M. Smith C.J. Spence J. Spitz D.R. Enhancement of carboplatin-mediated lung cancer cell killing by simultaneous disruption of glutathione and thioredoxin metabolism.Clin Cancer Res. 2011; 17: 6206-6217Crossref PubMed Scopus (80) Google Scholar]. Whether this strategy would be effective in malignant hematopoietic cells, specifically malignant B cells, has not yet been investigated, to our knowledge.Increasing cellular oxidative stress can induce cancer cell death directly as a result of the irreversible damage that reactive oxygen species elicit on cellular lipids, DNA, and/or proteins. Oxidative stress can also induce cancer cell death indirectly by modulating numerous redox-dependent cellular pathways that mediate cell survival. One such redox-dependent regulatory mechanism involves the posttranslational modification of reduced cysteines (or thiols) on proteins involved in diverse cellular functions, a process known as thiol-based redox switching [18Antelmann H. Helmann J.D. Thiol-based redox switches and gene regulation.Antioxid Redox Signal. 2011; 14: 1049-1063Crossref PubMed Scopus (273) Google Scholar]. The nuclear factor κB (NF-κB) signaling pathway, a pivotal cellular prosurvival pathway that is often dysregulated in cancer, is regulated in part by thiol-based redox switching. Notably, AUR inhibits the nuclear translocation, and thus activation, of NF-κB by inhibiting the activation of the inhibitory κB kinase (IKK) complex through its modification of cysteine 779 on IKK [19Jeon K.I. Jeong J.Y. Jue D.M. Thiol-reactive metal compounds inhibit NF-kappa B activation by blocking I kappa B kinase.J Immunol. 2000; 164: 5981-5989Crossref PubMed Scopus (195) Google Scholar, 20Jeon K.I. Byun M.S. Jue D.M. Gold compound auranofin inhibits IkappaB kinase (IKK) by modifying Cys-179 of IKKbeta subunit.Exp Mol Med. 2003; 35: 61-66Crossref PubMed Scopus (132) Google Scholar].In addition to inhibiting NF-κB activation, AUR is thought to inhibit TrxR activation via modification of cysteine and selonocysteine residues (thiols) in the redox centers of TrxR. Thus, given that BSO results in GSH depletion and that GSH reduces free thiols, we hypothesized that BSO would augment the inhibitory effect of AUR on both TrxR activity and NF-κB activation. Further, we hypothesized that the cytotoxicity elicited by the combination of AUR and BSO was due, in part, to inhibition of both TrxR and NF-κB.Here, we show that mantle cell lymphoma (MCL) cells exhibit dramatically reduced viability following combined exposure to AUR and BSO, even at low concentrations of both agents. Similar effects were observed for both diffuse large B-cell lymphoma (DLBCL) and MM cells; however, normal B-cell viability was less affected. Notably, BSO augmented AUR-induced inhibition of both TrxR and NF-κB signaling. Finally, the cytotoxic effects of AUR and BSO were attenuated by overexpressing either TrxR or NF-κB proteins, supporting a significant functional role for inhibition of these signaling pathways in the AUR+BSO synergistic toxicity. Collectively, these findings support the notion that dual targeting of cellular antioxidant systems may exert selective toxicity toward malignant B cells.Materials and methodsCell culture and primary cellsThe human DLBCL cell lines SUD-HL6 and OCI-LY10, the MCL cell lines Rec-1 and Granta, and the MM cell lines U266 and KMS-12-PE were cultured as previously described [21Namba M. Ohtsuki T. Mori M. et al.Establishment of five human myeloma cell lines.Vitro Cell Dev Biol. 1989; 25: 723-729Crossref PubMed Scopus (60) Google Scholar, 22Sniderhan L.F. Garcia-Bates T.M. Burgart M. Bernstein S.H. Phipps R.P. Maggirwar S.B. Neurotrophin signaling through tropomyosin receptor kinases contributes to survival and proliferation of nonHodgkin lymphoma.Exp Hematol. 2009; 37: 1295-1309Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 23Skalska J. Brookes P.S. Nadtochiy S.M. et al.Modulation of cell surface protein free thiols: A potential novel mechanism of action of the sesquiterpene lactone parthenolide.PLoS One. 2009; 4: e8115Crossref PubMed Scopus (55) Google Scholar, 24Dai Y. Landowski T.H. Rosen S.T. Dent P. Grant S. Combined treatment with the checkpoint abrogator UCN-01 and MEK1/2 inhibitors potently induces apoptosis in drug-sensitive and -resistant myeloma cells through an IL-6-independent mechanism.Blood. 2002; 100: 3333-3343Crossref PubMed Scopus (85) Google Scholar].B cells were isolated from fresh peripheral blood mononuclear cells from one healthy donor by negative magnetic bead selection of non–B cells, according to the manufacturer's protocol (Miltenyi Biotec, Auburn, CA). The donor provided signed, written, informed consent. All procedures and methods were approved by the Research Subjects Review Board at the University of Rochester Medical Center.Primary patient-derived MCL cells were obtained from the University of Rochester Medical Center Tissue Core under an Institutional Review Board–approved protocol. These tissue specimens were fully anonymous; hence patient demographics are not available.Chemical reagentsAll chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.Cell viability assaysCells were treated with varying concentrations of AUR or BSO, alone or together, for 24 hours, and a MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was performed as described previously [23Skalska J. Brookes P.S. Nadtochiy S.M. et al.Modulation of cell surface protein free thiols: A potential novel mechanism of action of the sesquiterpene lactone parthenolide.PLoS One. 2009; 4: e8115Crossref PubMed Scopus (55) Google Scholar]. The alamarBlue cell viability assay (Life Technologies, Grand Island, NY) was used according to the manufacturer's protocol to assess the viability of primary cells. The Annexin V-FITC Apoptosis Detection kit (BD Biosciences, San Jose, CA) was also used according to the manufacturer's instructions.Thioredoxin reductase activityTo measure TrxR activity, Granta cells were incubated with 100 nmol/L AUR or 5 μmol/L BSO, alone or in combination, for 18 hours. Subsequently, TrxR activity was measured spectrophotometrically using a kit, according to the manufacturer's protocol (Sigma-Aldrich).Electrophoretic mobility shift assayGranta cells were treated with the indicated concentrations of AUR and BSO, alone or in combination, for 6 hours. Following these treatments, nuclear extracts were prepared as described previously [25Schreiber E. Matthias P. Muller M.M. Schaffner W. Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells.Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3908) Google Scholar, 26Ramirez S.H. Sanchez J.F. Dimitri C.A. Gelbard H.A. Dewhurst S. Maggirwar S.B. Neurotrophins prevent HIV Tat-induced neuronal apoptosis via a nuclear factor-kappaB (NF-kappaB)-dependent mechanism.J Neurochem. 2001; 78: 874-889Crossref PubMed Scopus (82) Google Scholar, 27Maggirwar S.B. Ramirez S. Tong N. Gelbard H.A. Dewhurst S. Functional interplay between nuclear factor-kappaB and c-Jun integrated by coactivator p300 determines the survival of nerve growth factor-dependent PC12 cells.J Neurochem. 2000; 74: 527-539Crossref PubMed Scopus (39) Google Scholar]. Electrophoretic mobility shift assays were performed by incubating nuclear extracts with IR-Dye-700-conjugated, double-stranded DNA probe at room temperature for 10 min, or an additional 10 min incubation with NF-κB-specific antibodies for supershift analysis, followed by resolution of the complexes on native 4% polyacrylamide gels. The bands were then visualized using images acquired on an Odyssey infrared imager (LI-COR Biosciences, Lincoln, NE). The double-stranded oligonucleotide probe is as follows (upper strand): 5′/5IRD700/CAACGGCAGGGGAATTCCCCTCTCCTT-3′.Luciferase assayLuciferase reporter plasmids containing either the NF-κB- or OCT-1-responsive elements upstream of a firefly luciferase gene were transfected into Granta cells, together with a vector expressing IκBαS1/2 or an empty vector, using Nucleofector (Amaxa/Lonza, Basel, Switzerland). The total amount of plasmid DNA was kept constant at 5 μg for each transfection. Thirty minutes after the transfection, cells were plated into single wells and were left untreated or incubated for 6 hours with the indicated doses of AUR or BSO, alone or in combination. Cell lysates were prepared using reporter lysis buffer, and luciferase activity was measured with a SpectraMax M3 plate reader (Molecular Devices, Sunnyvale, CA). In these assays, total protein amount as determined by Bradford assay was used to normalize the samples, as a single transfection was divided into multiple wells for subsequent treatments.Immunoblot analysesFollowing the indicated manipulations, whole-cell lysates were prepared in E lysis buffer [28Kiebala M. Polesskaya O. Yao Z. Perry S.W. Maggirwar S.B. Nuclear factor-kappa B family member RelB inhibits human immunodeficiency virus-1 Tat-induced tumor necrosis factor-alpha production.PLoS One. 2010; 5: e11875Crossref PubMed Scopus (27) Google Scholar]. Lysates were fractionated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, and protein was electrophoretically transferred to Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (GE Healthcare Bio-Sciences Corporation, Piscataway, NJ). The membranes were analyzed for immunoreactivity with primary antibodies raised against RelA (1:1000), RelB (1:1000), IκBα (1:1000), or α-Tubulin (1:1000; all from Santa Cruz Biotechnologies, Santa Cruz, CA), TrxR, p100/p52, or actin (1:1000; from Cell Signaling Technology, Danvers, MA). Bound antibodies were detected by species-specific, IRDye-conjugated secondary antibodies (1:20,000; LI-COR Biosciences). The membranes were then subjected to Odyssey infrared imaging (LI-COR Biosciences, Lincoln, NE).Quantitative real-time polymerase chain reaction analysisB-cell lymphoma-2-like protein 1 (Bcl-xL) mRNA levels were determined via quantitative real-time polymerase chain reaction (qRT-PCR) using the following primers: Bcl-xL F 5′-GATCCCCATGGCAGCAGTAAAGCAAG-3′, R 5′-CCCCATCCCGGAAGAGTTCATTCACT-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) F 5′-AGGTGAAGGTCGGAGTCA-3′, R5′-GGTCATTGATGGCAACAA-3′. Levels of mRNA were compared using the ΔΔCt method.Adenovirus transductionWe transduced U266 cells with recombinant adenovirus vectors that express green fluorescent protein (GFP) and RelA [26Ramirez S.H. Sanchez J.F. Dimitri C.A. Gelbard H.A. Dewhurst S. Maggirwar S.B. Neurotrophins prevent HIV Tat-induced neuronal apoptosis via a nuclear factor-kappaB (NF-kappaB)-dependent mechanism.J Neurochem. 2001; 78: 874-889Crossref PubMed Scopus (82) Google Scholar], RelB (Cell Biolabs, San Diego, CA), or an empty control vector (Cell Biolabs; biosafety level 2). Vectors were constructed and propagated using previously described methods [29He T.C. Zhou S. da Costa L.T. Yu J. Kinzler K.W. Vogelstein B. A simplified system for generating recombinant adenoviruses.Proc Natl Acad Sci U S A. 1998; 95: 2509-2514Crossref PubMed Scopus (3230) Google Scholar], and they were handled following standard biosafety level 2 procedures. The viral vectors were used to transduce U266 cells at a multiplicity of infection of 100. Between 18 and 24 hours posttransduction, cells were treated with AUR or BSO, alone or in combination, for an additional 24 hours.Statistical analysesSynergy was analyzed at each testable dose combination using Laska et al.'s model-free test of synergy [30Laska E.M. Meisner M. Siegel C. Simple designs and model-free tests for synergy.Biometrics. 1994; 50: 834-841Crossref PubMed Scopus (42) Google Scholar] with unequal-variance t tests (in R software, http://www.r-project.org) as the building blocks. Adjustment for multiple comparisons over dose combinations via control of the false discovery rate was considered but deemed unnecessary, given that the majority of the synergy p values were significant and that they always clustered in contiguous synergistic dose combination regions.Mean data values and the standard error of the mean were calculated for each variable. One-way analysis of variance, followed by Bonferroni's test for multiple comparisons, was used to analyze data involving the analysis of multiple sample groups. A value of p < 0.05 was designated as statistically significant (Prism 4.0 software; GraphPad Software, La Jolla, CA).ResultsCombined toxicity of Auranofin and buthionine-sulfoximineThe effect of AUR in combination with BSO on the viability of various malignant B-cell lines was determined using an MTT assay following 24-hour exposure to these drugs, either alone or together. At low nanomolar concentrations, AUR alone caused minimal levels of cell death, and BSO alone was nontoxic in the MCL cell lines Granta and Rec-1. However, simultaneous exposure of the cells to both drugs dramatically enhanced the cytotoxicity, even at the lowest tested AUR concentration of 100 nmol/L (Fig. 1A). Similarly, BSO enhanced AUR-mediated toxicity in the DLBCL cell lines OCI-LY10 and SUDHL6 (Fig. 1B) and in the MM cell lines U266 and KMS-12-PE (Fig. 1C). Laska et al.'s model-free test of synergy [30Laska E.M. Meisner M. Siegel C. Simple designs and model-free tests for synergy.Biometrics. 1994; 50: 834-841Crossref PubMed Scopus (42) Google Scholar] confirmed the synergistic action of these two drugs together on cancer cell death at the multiple-dose combinations that were tested. This method has previously been used to test the effects of drug combinations on cancer cell growth [31Dasmahapatra G. Lembersky D. Son M.P. et al.Obatoclax interacts synergistically with the irreversible proteasome inhibitor carfilzomib in GC- and ABC-DLBCL cells in vitro and in vivo.Mol Cancer Ther. 2012; 11: 1122-1132Crossref PubMed Scopus (26) Google Scholar, 32Lu G. Xiao H. You H. et al.Synergistic inhibition of lung tumorigenesis by a combination of green tea polyphenols and atorvastatin.Clin Cancer Res. 2008; 14: 4981-4988Crossref PubMed Scopus (63) Google Scholar, 33Liu T. Yacoub R. Taliaferro-Smith L.D. et al.Combinatorial effects of lapatinib and rapamycin in triple-negative breast cancer cells.Mol Cancer Ther. 2011; 10: 1460-1469Crossref PubMed Scopus (77) Google Scholar]. The most profound combined toxicity of these drugs occurred in Granta cells and U266 cells; hence, these two cell lines were used in subsequent experiments investigating potential mechanisms underlying this synergism.We performed Annexin V/propidium iodide (PI) staining in Granta cells at multiple time points following treatment to determine the mechanism of AUR+BSO-induced cell death. At 6 hours posttreatment, there were no changes in levels of Annexin V single-positive (early apoptotic) or Annexin V/PI double-positive (late apoptotic/dead) cell populations. However, at both 18 hours (data not shown) and 24 hours (Fig. 1D) posttreatment, there was a large increase in the Annexin V/PI double-positive (late apoptotic/dead) cell population, thus suggesting that AUR+BSO may be directly inducing necrotic cell death in Granta cells. This is consistent with the report by Sobhakumari et al. [16Sobhakumari A. Love-Homan L. Fletcher E.V. et al.Susceptibility of human head and neck cancer cells to combined inhibition of glutathione and thioredoxin metabolism.PLoS One. 2012; 7: e48175Crossref PubMed Scopus (55) Google Scholar], where the researchers suggest that AUR+BSO induces necrosis in human head and neck cancer cells.Enhanced lethality involves inhibition of TrxR activityAuranofin is a well-known inhibitor of the Trx antioxidant system through inhibition of mitochondrial TrxR [34Cox A.G. Brown K.K. Arner E.S. Hampton M.B. The thioredoxin reductase inhibitor auranofin triggers apoptosis through a Bax/Bak-dependent process that involves peroxiredoxin 3 oxidation.Biochem Pharmacol. 2008; 76: 1097-1109Crossref PubMed Scopus (125) Google Scholar, 35Rigobello M.P. Bindoli A. Mitochondrial thioredoxin reductase purification, inhibitor studies, and role in cell signaling.Methods Enzymol. 2010; 474: 109-122Crossref PubMed Scopus (41) Google Scholar, 36Rigobello M.P. Callegaro M.T. Barzon E. Benetti M. Bindoli A. Purification of mitochondrial thioredoxin reductase and its involvement in the redox regulation of membrane permeability.Free Radic Biol Med. 1998; 24: 370-376Crossref PubMed Scopus (119) Google Scholar, 37Rigobello M.P. Folda A. Baldoin M.C. Scutari G. Bindoli A. Effect of auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase.Free Radic Res. 2005; 39: 687-695Crossref PubMed Scopus (81) Google Scholar, 38Liu J.J. Liu Q. Wei H.L. Yi J. Zhao H.S. Gao L.P. Inhibition of thioredoxin reductase by auranofin induces apoptosis in adriamycin-resistant human K562 chronic myeloid leukemia cells.Pharmazie. 2011; 66: 440-444PubMed Google Scholar]. As expected, we observed a significant reduction in TrxR activity after 18 hours of treatment with AUR in Granta cells. Interestingly, TrxR activity was further reduced following exposure to AUR and BSO in combination (Fig. 2A). The effect of TrxR overexpression on AUR+BSO-mediated cell death was next determined using an MTT assay in U266 cells stably overexpressing either wild-type TrxR or mutated TrxR (mutTrxR), which was unable to reduce oxidized thioredoxin. The untransfected parent cell line U266 was used as a control. A higher level of TrxR expression in both the TrxR and mutTrxR stable cell lines was confirmed by immunoblot analysis (Fig. 2B). Auranofin alone caused minimal cell death, and this was not significantly altered with TrxR overexpression (Fig. 2B). When combined with 5 μmol/L BSO, AUR drastically decreased cell viability. Interestingly, at the two lowest AUR concentrations, TrxR overexpression significantly protected against AUR+BSO-mediated toxicity (Fig. 2C), thus confirming that AUR's inhibition of TrxR contributed to the cell death induced by this drug combination.Figure 2Effect of AUR and BSO on thioredoxin reductase activity. (A) Granta cells were treated with AUR or BSO, alone or together, at the indicated concentrations for 18 hours. Thioredoxin reducatase activity was measured using the Sigma Thioredoxin Reductase assay kit. Results are shown as percent activity compared with DMSO-treated control cells. (B,C) U266 cells either untransfected (control), or stably overexpressing mutant thioredoxin reductase (mutTrxR) or wild-type thioredoxin reductase (TrxR) were treated with the indicated concentrations of AUR, either (B) alone or (C) together with 5 μmol/L BSO for 24 hours. Cell viability was measured using the MTT assay. Percent survival was calculated as compared with DMSO-treated control cells. Immunoblot analysis was performed on whole-cell lysates from U266 cells, either untransfected (control) or stably overexpressing mutTrxR or wild-type TrxR. Overexpression of TrxR was confirmed using an anti-TrxR antibody. Actin was used as a loading control. Statistical significance is indicated as compared with untransfected control cells (***p < 0.001, **p < 0.01) or as compared with mutTrxR expressing cells (**p < 0.01, *p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Buthionine-sulfoximine augments AUR inhibition of nuclear factor-κB signalingThe survival of malignant cells is often linked to constitutive activation of the NF-κB signaling pathway [39Ben-Neriah Y. Karin M. Inflammation meets cancer, with NF-kappaB as the matchmaker.Nat Immunol. 2011; 12: 715-723Crossref PubMed Scopus (1120) Google Scholar]. Since AUR has been previously shown to inhibit NF-κB signaling [19Jeon K.I. Jeong J.Y. Jue D.M. Thiol-reactive metal compounds inhibit NF-kappa B activation by blocking I kappa B kinase.J
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