N-Acetyl-l-cysteine Enhances Apoptosis through Inhibition of Nuclear Factor-κB in Hypoxic Murine Embryonic Fibroblasts
2004; Elsevier BV; Volume: 279; Issue: 48 Linguagem: Inglês
10.1074/jbc.m406749200
ISSN1083-351X
AutoresSuparna Qanungo, Mi Wang, Anna‐Liisa Nieminen,
Tópico(s)Immune Response and Inflammation
ResumoIn this study, we investigated the role of reduced glutathione (GSH) and nuclear factor-κB (NFκB) in hypoxia-induced apoptosis. Hypoxia caused p53-dependent apoptosis in murine embryonic fibroblasts transfected with Ras and E1A. N-Acetyl-l-cysteine (NAC) but not other antioxidants, such as the vitamin E analog trolox and epigallocatechin-3-gallate, enhanced hypoxia-induced caspase-3 activation and apoptosis. NAC also enhanced hypoxia-induced apoptosis in two human cancer cell lines, MIA PaCa-2 pancreatic cancer cells and A549 lung carcinoma cells. In murine embryonic fibroblasts, all three antioxidants blocked hypoxia-induced reactive oxygen species formation. NAC did not enhance hypoxia-induced cytochrome c release but did enhance poly-(ADP ribose) polymerase cleavage, indicating that NAC acted at a post-mitochondrial level. NAC-mediated enhancement of apoptosis was mimicked by incubating cells with GSH monoester, which increased intracellular GSH similarly to NAC. Hypoxia promoted degradation of an inhibitor of κB(IκBα), NFκB-p65 translocation into the nucleus, NFκB binding to DNA, and subsequent transactivation of NFκB, which increased X chromosome-linked inhibitor of apoptosis protein levels. NAC failed to block degradation by IκBα and sequestration of the p65 subunit of NFκB to the nucleus. However, NAC did abrogate hypoxia-induced NFκB binding to DNA, NFκB-dependent gene expression, and induction of X chromosome-linked inhibitor of apoptosis protein. In conclusion, NAC enhanced hypoxic apoptosis by a mechanism apparently involving GSH-dependent suppression of NFκB transactivation. In this study, we investigated the role of reduced glutathione (GSH) and nuclear factor-κB (NFκB) in hypoxia-induced apoptosis. Hypoxia caused p53-dependent apoptosis in murine embryonic fibroblasts transfected with Ras and E1A. N-Acetyl-l-cysteine (NAC) but not other antioxidants, such as the vitamin E analog trolox and epigallocatechin-3-gallate, enhanced hypoxia-induced caspase-3 activation and apoptosis. NAC also enhanced hypoxia-induced apoptosis in two human cancer cell lines, MIA PaCa-2 pancreatic cancer cells and A549 lung carcinoma cells. In murine embryonic fibroblasts, all three antioxidants blocked hypoxia-induced reactive oxygen species formation. NAC did not enhance hypoxia-induced cytochrome c release but did enhance poly-(ADP ribose) polymerase cleavage, indicating that NAC acted at a post-mitochondrial level. NAC-mediated enhancement of apoptosis was mimicked by incubating cells with GSH monoester, which increased intracellular GSH similarly to NAC. Hypoxia promoted degradation of an inhibitor of κB(IκBα), NFκB-p65 translocation into the nucleus, NFκB binding to DNA, and subsequent transactivation of NFκB, which increased X chromosome-linked inhibitor of apoptosis protein levels. NAC failed to block degradation by IκBα and sequestration of the p65 subunit of NFκB to the nucleus. However, NAC did abrogate hypoxia-induced NFκB binding to DNA, NFκB-dependent gene expression, and induction of X chromosome-linked inhibitor of apoptosis protein. In conclusion, NAC enhanced hypoxic apoptosis by a mechanism apparently involving GSH-dependent suppression of NFκB transactivation. Hypoxia is a frequent feature of rapidly growing malignant tumors and their metastases. Tissue hypoxia resulting from inadequate blood supply generally occurs at early stages of tumor development, beginning at a tumor diameter of only a few millimeters (1Helmlinger G. Yuan F. Dellian M. Jain R.K. Nat. Med. 1997; 3: 177-182Crossref PubMed Scopus (1316) Google Scholar). In solid tumors, hypoxia causes a selection of mutations that makes cells resistant to apoptosis and less responsive to cancer therapy (2Schmaltz C. Hardenbergh P.H. Wells A. Fisher D.E. Mol. Cell. Biol. 1998; 18: 2845-2854Crossref PubMed Scopus (193) Google Scholar, 3Graeber T.G. Osmanian C. Jacks T. Housman D.E. Koch C.J. Lowe S.W. Giaccia A.J. Nature. 1996; 379: 88-91Crossref PubMed Scopus (2153) Google Scholar). Hypoxia also induces genes that protect cells against apoptosis. One of these genes is nuclear factor-κB (NFκB). 1The abbreviations used are: NFκB, nuclear factor-κB; IκB, inhibitor of nuclear factor κB; IKK, IκB kinase; c-FLIP, caspase-8-FADD-like IL-1β-converting enzyme inhibitory protein; XIAP, X chromosome-linked inhibitor of apoptosis protein; ROS, reactive oxygen species; NAC, N-acetyl-l-cysteine; d-NAC, N-acetyl-d-cysteine; GSH, reduced glutathione; MAPK, mitogen activated protein kinase; PARP, poly(ADP ribose) polymerase; MEF, murine embryonic fibroblast; CM-H2DCFDA, 2′,7′-dichlorodihydrofluorescin diacetate; DCF, dichlorofluorescein; EGCG, epigallocatechin-3-gallate; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay.1The abbreviations used are: NFκB, nuclear factor-κB; IκB, inhibitor of nuclear factor κB; IKK, IκB kinase; c-FLIP, caspase-8-FADD-like IL-1β-converting enzyme inhibitory protein; XIAP, X chromosome-linked inhibitor of apoptosis protein; ROS, reactive oxygen species; NAC, N-acetyl-l-cysteine; d-NAC, N-acetyl-d-cysteine; GSH, reduced glutathione; MAPK, mitogen activated protein kinase; PARP, poly(ADP ribose) polymerase; MEF, murine embryonic fibroblast; CM-H2DCFDA, 2′,7′-dichlorodihydrofluorescin diacetate; DCF, dichlorofluorescein; EGCG, epigallocatechin-3-gallate; DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent assay. NFκB is a critical transcription factor that tips the balance from apoptosis of cells to proliferation and malignant growth of tumor cells. NFκB belongs to the Re1 family, which includes five mammalian Re1/NFκB proteins: Re1A (p65), c-Re1, Re1B, NFκB1 (p50/p105), and NFκB2 (p52/p100) (4Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2422) Google Scholar). The activity of NFκB is regulated by movement between the cytoplasm and nucleus in response to cell stimulation (5Greten F.R. Karin M. Cancer Lett. 2004; 206: 193-199Crossref PubMed Scopus (354) Google Scholar). NFκB dimers containing Re1A or c-Re1 are retained in the cytoplasm through interaction with inhibitor of κB(IκB) repressor proteins (IκBα, IκBβ, IκBγ, and IκBϵ) (6Fan C. Li Q. Ross D. Engelhardt J.F. J. Biol. Chem. 2003; 278: 2072-2080Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Potent NFκB activators, such as tumor necrosis factor-α, interleukin-1, and lipopolysaccharide, cause IκB kinase (IKK)-mediated phosphorylation of IκBα on serine 32 and serine 36 residues, followed by ubiquitination at the nearby lysine residues and proteasomal degradation (7Lin A. Karin M. Semin. Cancer Biol. 2003; 13: 107-114Crossref PubMed Scopus (345) Google Scholar). This modification of IκBα exposes the nuclear localization signal of NFκB and leads to NFκB translocation to the nucleus, presumably as a consequence of binding to karyopherins (8Rothwarf D.M. Karin M. Sci. STKE. 1999; 1999: RE1Crossref PubMed Google Scholar), proteins responsible for nucleocytoplasmic transport in cells. An alternative, less-characterized pathway of NFκB activation is by tyrosine 42 phosphorylation of IκBα after hypoxia/reoxygenation or pervanadate treatment (6Fan C. Li Q. Ross D. Engelhardt J.F. J. Biol. Chem. 2003; 278: 2072-2080Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). However, in T-cells and bone marrow macrophages, tyrosine phosphorylation of IκBα is insufficient to activate NFκB, suggesting that tyrosine and serine kinases act at multiple levels to dissociate the IκBα/NFκB complex (8Rothwarf D.M. Karin M. Sci. STKE. 1999; 1999: RE1Crossref PubMed Google Scholar). Once IκBα is inactivated, NFκB dimers are free to localize to the nucleus, where they undergo further modification, mostly through phosphorylation of the Re1 proteins (9Sizemore N. Lerner N. Dombrowski N. Sakurai H. Stark G.R. J. Biol. Chem. 2002; 277: 3863-3869Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Activated NFκB in the nucleus binds to promoters of its target genes to initiate the transcription of several genes involved in immune responses, inflammation, viral infection, and cell survival (7Lin A. Karin M. Semin. Cancer Biol. 2003; 13: 107-114Crossref PubMed Scopus (345) Google Scholar, 10Bose S. Kar N. Maitra R. DiDonato J.A. Banerjee A.K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10890-10895Crossref PubMed Scopus (64) Google Scholar). NFκB suppresses apoptosis by inducing expression of various anti-apoptotic genes, including Bcl-xL, Bcl-2, hematopoietic-specific Bcl-2 homolog A1 (Bfl1) (11Wang C.Y. Guttridge D.C. Mayo M.W. Baldwin Jr., A.S. Mol. Cell. Biol. 1999; 19: 5923-5929Crossref PubMed Scopus (535) Google Scholar), caspase-8-FADD-like interleukin-1β-converting enzyme inhibitory protein (c-FLIP), tumor necrosis factor receptor-associated factor 1 (TRAF1), tumor necrosis factor receptor-associated factor 2 (TRAF2), and cellular inhibitors of apoptosis (c-IAPs) (7Lin A. Karin M. Semin. Cancer Biol. 2003; 13: 107-114Crossref PubMed Scopus (345) Google Scholar). NFκB also regulates X chromosome-linked IAP (XIAP/hILP), which inhibits caspase-3 and caspase-7 and prevents activation of pro-caspase-9 (12Deveraux Q.L. Leo E. Stennicke H.R. Welsh K. Salvesen G.S. Reed J.C. EMBO J. 1999; 18: 5242-5251Crossref PubMed Scopus (674) Google Scholar). Hypoxia promotes reactive oxygen species (ROS) formation in a number of cell types (13Dawson T.L. Gores G.J. Nieminen A.L. Herman B. Lemasters J.J. Am. J. Physiol. 1993; 264: C961-C967Crossref PubMed Google Scholar, 14Duranteau J. Chandel N.S. Kulisz A. Shao Z. Schumacker P.T. J. Biol. Chem. 1998; 273: 11619-11624Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 15Mansfield K.D. Simon M.C. Keith B. J. Appl. Physiol. 2004; 97: 1358-1366Crossref PubMed Scopus (84) Google Scholar). ROS, such as H2O2, may act as second messengers activating NFκB (16Schreck R. Albermann K. Baeuerle P.A. Free Radic. Res. Commun. 1992; 17: 221-237Crossref PubMed Scopus (1292) Google Scholar). Hyperoxia, which also induces ROS generation, causes nuclear translocation and activation of NFκB (17Cazals V. Nabeyrat E. Corroyer S. de Keyzer Y. Clement A. Biochim. Biophys. Acta. 1999; 1448: 349-362Crossref PubMed Scopus (43) Google Scholar). Conversely, increased cellular antioxidants prevent NFκB activation by cytokines (18Renard P. Zachary M.D. Bougelet C. Mirault M.E. Haegeman G. Remacle J. Raes M. Biochem. Pharmacol. 1997; 53: 149-160Crossref PubMed Scopus (71) Google Scholar). Although substantial evidence supports a role of ROS in NFκB activation, the contribution of redox regulation to NFκB activation is still controversial (19Hayakawa M. Miyashita H. Sakamoto I. Kitagawa M. Tanaka H. Yasuda H. Karin M. Kikugawa K. EMBO J. 2003; 22: 3356-3366Crossref PubMed Scopus (360) Google Scholar). N-Acetyl-l-cysteine (NAC) is a widely used thiol-containing antioxidant that is a precursor of reduced glutathione (GSH). GSH scavenges ROS in cells by interacting with OH· and H2O2, thus affecting ROS-mediated signaling pathways. NAC facilitates interleukin-1β-induced inducible nitric oxide synthase expression through an oxidation/reduction-related mechanism potentiating cytokine activation of the p44/42 MAPK signaling cascade (20Jiang B. Brecher P. Hypertension. 2000; 35: 914-918Crossref PubMed Scopus (32) Google Scholar, 21Kamata H. Manabe T. Kakuta J. Oka S. Hirata H. Ann. N. Y. Acad. Sci. 2002; 973: 419-422Crossref PubMed Scopus (25) Google Scholar). NAC inhibits activation of c-Jun N-terminal kinase, p38 MAPK, redox-sensitive activator protein-1, and NFκB transcription factor activities regulating the expression of numerous genes (21Kamata H. Manabe T. Kakuta J. Oka S. Hirata H. Ann. N. Y. Acad. Sci. 2002; 973: 419-422Crossref PubMed Scopus (25) Google Scholar, 22Zafarullah M. Li W.Q. Sylvester J. Ahmad M. Cell Mol. Life Sci. 2003; 60: 6-20Crossref PubMed Scopus (1039) Google Scholar). Although several reports show that NAC protects against oxidative stress-induced cell death (23Park S.A. Choi K.S. Bang J.H. Huh K. Kim S.U. J. Neurochem. 2000; 75: 946-953Crossref PubMed Scopus (81) Google Scholar, 24van Zandwijk N. Chest. 1995; 107: 1437-1441Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), other reports document that NAC can also induce apoptosis in colon carcinoma cells by increasing the pro-apoptotic Bax gene expression and by enhancing the susceptibility to the chemotherapeutic agent 5-fluorouracil (22Zafarullah M. Li W.Q. Sylvester J. Ahmad M. Cell Mol. Life Sci. 2003; 60: 6-20Crossref PubMed Scopus (1039) Google Scholar). Despite evidence that ROS play a role in hypoxia-induced apoptosis, the exact mechanisms of apoptotic death are not well understood. Therefore, the aim of this study was to establish causal links between GSH, NFκB activation, and cell survival in hypoxic transformed cells. We show that (i) NAC potentiates apoptosis of hypoxic transformed cells, and (ii) NAC inhibits NFκB-dependent expression of antiapoptotic proteins such as XIAP. Chemicals and Reagents—Monoclonal anti-NFκB p65, polyclonal anti-IκBα, anti-actin, anti-c-FLIP, and secondary horseradish peroxidase-linked antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Bcl-xL antibody was from Cell Signaling Technologies (Beverly, MA). Monoclonal antibodies against poly(ADP ribose) polymerase (PARP), cytochrome c, F1α-ATPase, XIAP, and Bcl-2 were purchased from Trevigen (Gaithersburg, MD), PharMingen (San Diego, CA), Molecular Probes (Eugene, OR), and BD Transduction Labs (San Jose, CA), respectively. Hoescht 33342 and Alexa 488-conjugated goat anti-mouse IgG were from Molecular Probes. α-Methylomuralide was obtained from Calbiochem (La Jolla, CA). The luciferase reporter construct pNFκB-Luc was a gift from Dr. Santanu Bose (Cleveland Clinic Foundation, Cleveland, OH), and N-acetyl-d-cysteine (d-NAC) was a gift from Dr. Ian Cotgreave (AstraZeneca, Stockholm, Sweden). All other chemicals were purchased from Sigma. Cell Culture—p53+/+ and p53-/- murine embryonic fibroblasts (MEFs) transformed with proto-oncogenes Ras and E1A were kindly provided by Dr. Scott Lowe (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). MEFs, MIA PaCa-2 human pancreatic cancer cells, and A549 human lung carcinoma cells were grown in complete culture medium containing Dulbecco's Modified Eagle's Medium supplemented with 25 mm glucose, 10% fetal bovine serum, and antibiotics (50 units/ml of penicillin and 50 μg/ml of streptomycin) in a humidified atmosphere of 5% CO2 and 95% air. MEFs were cultured on Primaria Petri dishes coated with 0.1% gelatin (Sigma). MIA PaCa-2 and A549 cells were cultured on Petri dishes without gelatin coating. For all experiments, cells were grown to 60–70% confluence. Hypoxia Experiments—MEFs, MIA PaCa-2, and A549 cells were cultured on 60-mm Petri dishes (1 × 106 cells/dish) before exposure to hypoxia. Subsequently, cells were preincubated with NAC for 1 h in complete culture medium supplemented with 10 mm glucose. Petri dishes were placed in hypoxic Plexiglass chambers (Billups-Rottenberg, Del Mar, CA) and sparged with 95% N2/5% CO2 at 2 psi for 4 min. The remaining final oxygen content was estimated to be ∼0.2% because of the presence of some residual oxygen. Chambers were then kept at 37 °C throughout the course of the experiment. Transfection and Reporter Assay—MEFs (1 × 106 cells/dish) were plated on 60-mm Petri dishes for 24 h. Subsequently, cells were transiently transfected with 1 μg of plasmid containing 2× NFκB promoter fused to the firefly luciferase gene (10Bose S. Kar N. Maitra R. DiDonato J.A. Banerjee A.K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10890-10895Crossref PubMed Scopus (64) Google Scholar) or 1 μg of empty vector luciferase construct using LipofectAMINE 2000 (Invitrogen). To normalize the transfection efficiency, cells were co-transfected with 4 μg of β-galactosidase control vector. Sixteen h after transfection, cells were exposed to hypoxia or normoxia for 4 h and then harvested. Relative luciferase activity was measured with a luciferase assay system (Promega, Madison, WI) using a luminometer (Turner Design). Luciferase activity was normalized for transfection efficiency using β-galactosidase activity, which was measured using a FluoReporter LacZ/β-galactosidase quantitation kit (Molecular Probes) according to the manufacturer's instructions. Luciferase activity was assessed as relative light units and used as an indicator of transcriptional induction of NFκB. Caspase-3 Activation—Caspase-3 activity was measured using a commercial kit (R&D Systems, Minneapolis, MN). Briefly, at the indicated time points, cultured MEFs were collected into a test tube, followed by centrifugation. The pellet was resuspended in a lysis buffer provided by the kit. Cell lysates were incubated with a caspase-3 fluorogenic substrate, DEVD-AFC (50 μm). Caspase-3 cleaves DEVD-AFC into free AFC, the fluorescence of which was measured with a CytoFluor 2000 fluorescence plate reader using 400-nm excitation and 505-nm emission light. Caspase-3 enzymatic activity was expressed as pmol/mg/min AFC formed. ROS Formation—Cultured cells were loaded with 10 μm 2′,7′-dichlorodihydrofluorescin diacetate (CM-H2DCFDA, Molecular Probes) for 1 h before exposure to hypoxia. Inside cells, esterases cleave the acetate esters to release free CM-H2DCF, which is non-fluorescent. After oxidation by ROS, CM-H2DCF is converted to green-fluorescing dichlorofluorescein (DCF) (25Nieminen A.L. Byrne A.M. Herman B. Lemasters J.J. Am. J. Physiol. 1997; 272: C1286-C1294Crossref PubMed Google Scholar). After hypoxia, medium containing CM-H2DCFDA was replaced, and cells were fixed and permeabilized with phosphate-buffered saline (PBS) containing 20% ethanol and 0.1% Tween 20. Subsequently, the cell extracts were centrifuged, and supernatants were collected. DCF fluorescence was measured with a fluorescence plate reader using 450-nm excitation and 530-nm emission filters. Measurement of GSH—GSH was measured by high-performance liquid chromatography (Hewlett-Packard series 1050). MEFs were washed with PBS and lysed using two freeze-thaw cycles. Cells were then scraped, suspended in buffer containing 0.1 m sodium phosphate, 5 mm EDTA, pH 8.0, and centrifuged. Supernatants were mixed with metaphosphoric acid to a final concentration of 1% to precipitate protein. After centrifugation at 12,000 × g for 15 min, supernatants were diluted (1:100) in a mobile phase containing 10 mm trichloroacetic acid and 69 mm monochloroacetic acid (pH adjusted to 2.72 with NaOH). GSH was resolved on a C18 reverse phase column (Vydac) and detected using electrochemical detection. A standard curve was generated using pure GSH, and results were calculated as nmol GSH/mg of protein (26Nulton-Persson A.C. Starke D.W. Mieyal J.J. Szweda L.I. Biochemistry. 2003; 42: 4235-4242Crossref PubMed Scopus (146) Google Scholar). Western Blot Analysis—At the indicated time points, total cell extracts were prepared in ice-cold RIPA lysis buffer (150 mm NaCl, 1 mm EGTA, 1% sodium deoxycholate, 1% Triton X-100, and 50 mm Tris-Cl, pH 8.0) supplemented with a mixture of protease inhibitors (Roche Diagnostics, Indianapolis, IN). The lysates were centrifuged, and the resulting supernatants were assayed for total protein content (Bio-Rad, Hercules, CA). Equivalent amounts of protein were diluted in sample buffer (200 mm Tris-Cl, 15% glycerol, 10% SDS, 5% β-mercaptoethanol, and 0.01% bromphenol blue, pH 6.8) and resolved on SDS-PAGE gel. The proteins were then transferred and immobilized onto polyvinylidene difluoride membranes (Millipore, Bedford, MA) and probed with appropriate primary and secondary antibodies. Immunodetection was accomplished by an enhanced chemiluminescence detection system (Pierce). Immunocytochemistry—At the indicated time points, MEFs cultured on gelatin-coated plastic coverslips were fixed with 3.7% formaldehyde for 30 min at room temperature. Subsequently, cells were rinsed with PBS and permeabilized with 0.25% Triton X-100 for 5 min. After two washes with PBS, cells were incubated with blocking solution (1% bovine serum albumin and 0.1% Tween 20 in PBS) for 1 h, followed by primary monoclonal antibody against p65 NFκB (1:100) overnight at 4 °C. Cells were then washed three times with PBS and incubated with anti-mouse secondary Alexafluor 488 (1:200) and again washed three times with PBS. Hoechst 33342 (0.5 μg/ml) was included in the final wash to stain the nuclei. Coverslips were attached on slides, mounting medium (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added, and the preparation was covered with a glass coverslip. Images of Alexafluor 488 fluorescence were collected using 488-nm excitation and >505-nm emission filters. Hoeschst fluorescence was imaged with a 720-nm excitation light from a two-photon Mira 900 laser and >420-nm emission filter using a Zeiss 510 NLO laser scanning confocal microscope. Cytosolic, Mitochondrial, and Nuclear Extracts—Nuclear and cytosolic extracts were prepared according to a modified method by Wang et al. (27Wang G.L. Jiang B.H. Semenza G.L. Biochem. Biophys. Res. Commun. 1995; 212: 550-556Crossref PubMed Scopus (158) Google Scholar). Briefly, cells cultured on 150-mm Petri dishes were scraped into ice-cold phosphate-buffered saline, centrifuged, and washed with five packed cell volumes of buffer A (250 mm sucrose, 20 mmN-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid, 10 mm KCl, 1.5 mm MgCl2, and 1 mm EDTA, pH 7.5). Cells were then resuspended in three to four packed cell volumes of buffer A and homogenized in a glass Dounce homogenizer. Nuclei were pelleted by centrifugation at 10,000 × g for 10 min at 4 °C. Supernatants were mixed with 0.5 volume of buffer B (75 mm Tris-Cl, 20% v/v glycerol, 300 mm KCl, and 600 mm EDTA, pH 7.5) and clarified by centrifugation at 100,000 × g for 30 min at 4 °C to obtain the soluble cytoplasmic fraction (S-100). Nuclear pellets were then resuspended in buffer C (420 mm KCl, 20 mm Tris-Cl, 20% v/v glycerol, and 1.5 mm MgCl2, pH 7.5), tubes were nutated for 30 min at 4 °C and centrifuged at 16,000 × g, and the supernatants were saved as nuclear extracts. Isolation of highly enriched mitochondrial and cytosolic fractions was carried out using a commercial kit (BioVision, Mountain View, CA) by following the manufacturer's protocol, with slight modifications (28Qanungo S. Haldar S. Basu A. Neoplasia. 2003; 5: 367-374Crossref PubMed Google Scholar). NFκB Activation—NFκB activation was measured with a Trans-AM NFκB kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. In this assay, the oligonucleotide containing the NFκB consensus sequence (5′-GGGACTTTCC-3′) is immobilized onto a 96-well plate. Only the active form of NFκB in the cell extract specifically binds to this oligonucleotide. Epitopes on p50, p52, p65, c-Re1, and Re1B are accessible only when NFκB is bound to its target DNA. Nuclear extracts (10 μg) were added to the wells, followed by the primary antibody against p65 and the horseradish peroxidase-conjugated secondary antibody. The optical density was determined on an absorbance plate reader at 450 nm. Assessment of Cell Death—Apoptotic and necrotic cell death was assessed independently. At the indicated time points, floating cells were collected, and adherent cells were trypsinized, centrifuged, and resuspended in PBS containing 200 μm digitonin (Calbiochem) and 50 μm propidium iodide (Molecular Probes). Digitonin permeabilizes the plasma membrane and allows propidium iodide to enter cells and stain all nuclei. Staining nuclei with propidium iodide in the presence of digitonin is analogous to staining nuclei with cell-permeable dyes such as 4′,6-diamidino-2-phenylindole. Fragmented nuclei were scored as apoptotic and counted using a 40× microscope objective and a rhodamine filter set. Necrotic cell death was assessed by incubating cells with trypan blue. Trypan blue enters cells and stains nuclei when the plasma membrane permeability barrier fails, a hallmark of necrosis (29Nieminen A.L. Gores G.J. Wray B.E. Tanaka Y. Herman B. Lemasters J.J. Cell Calcium. 1988; 9: 237-246Crossref PubMed Scopus (106) Google Scholar). Round, trypan blue-stained nuclei were counted as necrotic. At least 100 cells were counted from three different microscopic fields for each sample. Results were expressed as the percentage of all cells that were apoptotic or necrotic. Additionally, apoptotic cell death was assessed with a Cell Death Detection ELISAPLUS kit (Roche Applied Science, Indianapolis, IN), according to the manufacturer's instructions. Endogenous endonucleases activated during apoptosis cleave double-stranded DNA to mono- and oligonucleosomes of 180-bp multiples, which the ELISA kit measures. DNA fragments were measured in the cytoplasmic fractions. Statistical Analysis—Data are presented as means ± S.E. from at least three independent experiments. Differences were assessed by two-tailed paired Student's t test with Instat Software (GraphPAD, San Diego, CA). p < 0.05 was considered to be statistically significant. NAC Enhances Hypoxia-induced Apoptosis—NAC is a nontoxic precursor of GSH and has been reported to protect cells against oxidative stress (22Zafarullah M. Li W.Q. Sylvester J. Ahmad M. Cell Mol. Life Sci. 2003; 60: 6-20Crossref PubMed Scopus (1039) Google Scholar). We exposed p53+/+ MEFs to hypoxia for 12 h in the presence and absence of NAC. Hypoxia alone caused 12% apoptosis compared with 2% during normoxia, as assessed by nuclear morphology typical of apoptosis (Fig. 1A). Apoptotic cells showed fragmented nuclei when stained with propidium iodide in the presence of digitonin (Fig. 1A, inset). NAC enhanced hypoxia-induced apoptosis in a dose- and time-dependent manner (Fig. 1, A and B). Apoptosis increased from 12% without NAC to 46% with 20 mm NAC (Fig. 1A). During normoxia, NAC increased apoptosis from 2.7% without NAC to 4.2% with 20 mm NAC (Fig. 1A). However, this increase of apoptosis was not dose-dependent. In contrast to p53+/+ MEFs, p53-/- MEFs were completely resistant to hypoxia and NAC (data not shown). In p53+/+ MEFs, NAC promoted hypoxia-induced apoptosis in a time-dependent manner. Apoptosis increased from 1.6, 2.8, 5.7, and 12.0% without NAC to 1.9, 7.9, 19.5, and 46.0% with 20 mm NAC after 0, 4, 7, and 12 h of hypoxia, respectively (Fig. 1B). In the subsequent experiments, we chose 20 mm NAC to characterize the cellular and molecular targets of NAC. This concentration was chosen because it maximally promoted apoptosis during hypoxia (Fig. 1A and data not shown). Furthermore, previous studies show that 20 to 30 mm NAC inhibits ROS formation and NFκB activation in cancer cells (19Hayakawa M. Miyashita H. Sakamoto I. Kitagawa M. Tanaka H. Yasuda H. Karin M. Kikugawa K. EMBO J. 2003; 22: 3356-3366Crossref PubMed Scopus (360) Google Scholar, 30Hammond E.M. Dorie M.J. Giaccia A.J. J. Biol. Chem. 2003; 278: 12207-12213Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). To further confirm that hypoxia-induced and hypoxia plus NAC-induced cell death was primarily apoptotic, histone-associated DNA fragments were measured in cytoplasmic fractions. Hypoxia alone increased DNA fragments 20% compared with normoxia, whereas hypoxia plus NAC increased DNA fragments by 260% compared with normoxia (Fig. 1C). During normoxia, however, NAC had no effect on DNA fragment formation. To assess necrotic cell death, MEFs were incubated with 0.2% trypan blue. Round nuclei stained with trypan blue were scored as necrotic. During normoxia, 11% of cells were necrotic (Fig. 1D). Hypoxia, hypoxia plus NAC, and NAC during normoxia did not increase necrosis significantly. The small percentage of necrosis observed in normoxic cells may be partly attributable to trypsinization before trypan blue staining and not related to experimental treatments. Together, our results indicate that cell death promoted by NAC was apoptotic. We also determined the effect of NAC on two human cancer cell lines. MIA PaCa-2 pancreatic cancer cells were exposed to hypoxia in the presence and absence of 20 mm NAC. NAC increased apoptosis from 9% without NAC to 30% with 20 mm NAC after 15 h of hypoxia (Fig. 1E). In A549 lung carcinoma cells, NAC increased apoptosis from 6% without NAC to 22% with 20 mm NAC after 26 h of hypoxia (Fig. 1F). NAC alone during normoxia caused a small increase of apoptosis in A549 cells but no increase in MIA PaCa-2 cells. NAC Does Not Accelerate Hypoxia-induced Cytochrome c Release—Next, we assessed whether NAC-enhanced apoptosis during hypoxia was mediated by accelerated cytochrome c release from mitochondria to the cytosol. After 12 h of exposure to hypoxia, a portion of cytochrome c was released from mitochondria to the cytosol (Fig. 2A). However, NAC did not change the extent of cytochrome c release, indicating that NAC-mediated enhancement of apoptosis acted downstream of mitochondria. Because NAC did not seem to enhance apoptosis through mitochondrial cytochrome c release, we determined whether post-mitochondrial events, such as PARP cleavage, were affected by NAC during hypoxia. PARP is a target for cysteine proteases, and 116-kDa PARP is frequently cleaved by caspase-3 into an 85-kDa fragment during apoptosis (31Qanungo S. Basu A. Das M. Haldar S. Oncogene. 2002; 21: 4149-4157Crossref PubMed Scopus (57) Google Scholar). After 7 and 14 h of hypoxia alone, we could not detect the cleaved 85-kDa fragment of PARP by Western blot analysis (Fig. 2B). In contrast, hypoxia in combination with NAC produced the 85-kDa PARP-degraded product (Fig. 2B). Collectively, the results indicate that enhancement of hypoxia-induced apoptotic cell death by NAC was not mediated by increased cytochrome c release but rather through some other downstream effector molecules that resulted in PARP cleavage. NAC Blocks Hypoxia-induced ROS Format
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