Glutathione Supplementation Potentiates Hypoxic Apoptosis by S-Glutathionylation of p65-NFκB
2007; Elsevier BV; Volume: 282; Issue: 25 Linguagem: Inglês
10.1074/jbc.m610934200
ISSN1083-351X
AutoresSuparna Qanungo, David W. Starke, Harish V. Pai, John J. Mieyal, Anna‐Liisa Nieminen,
Tópico(s)Sulfur Compounds in Biology
ResumoIn murine embryonic fibroblasts, N-acetyl-l-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFκB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFκB that are responsible for NFκB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFκB DNA binding and NFκB transactivation by 2.6- and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFκB protein levels and p65-NFκB nuclear translocation during hypoxia. Pharmacological inhibition of the NFκB pathway also induced hypoxic apoptosis, indicating that the NFκB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFκB to DNA, suggesting that most sulfhydryls in p65-NFκB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFκB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFκB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFκB activity. This finding indicated that p65-NFκB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFκB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFκB is most likely responsible for NAC-mediated NFκB inactivation and enhanced hypoxic apoptosis. In murine embryonic fibroblasts, N-acetyl-l-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFκB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFκB that are responsible for NFκB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFκB DNA binding and NFκB transactivation by 2.6- and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFκB protein levels and p65-NFκB nuclear translocation during hypoxia. Pharmacological inhibition of the NFκB pathway also induced hypoxic apoptosis, indicating that the NFκB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFκB to DNA, suggesting that most sulfhydryls in p65-NFκB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFκB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFκB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFκB activity. This finding indicated that p65-NFκB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFκB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFκB is most likely responsible for NAC-mediated NFκB inactivation and enhanced hypoxic apoptosis. Tumor hypoxia is strongly associated with tumor propagation, malignant progression, and resistance to chemoand radiation therapy (1Vaupel P. Mayer A. Transfus. Clin. Biol. 2005; 12: 5-10Crossref PubMed Scopus (121) Google Scholar). NFκB 2The abbreviations used are: NFκB, nuclear factor κB; BSA, bovine serum albumin; d-NAC, N-acetyl-d-cysteine; DTT, dithiothreitol; GRx, glutaredoxin; GRx-SSG·¯, GRx-S-S-glutathione disulfide anion radical; GS., glutathionyl thiol radical; IKK, IκB kinase; IκB, inhibitor of nuclear factorκB; MEFs, murine embryonic fibroblasts; NAC, N-acetyl-l-cysteine; NADPH, nicotinamide adenine dinucleotide phosphate; NEM, N-ethylmaleimide; O2·¯, superoxide; OH., hydroxyl radical; PARP, poly(ADP-ribose) polymerase; XIAP, X chromosome-linked inhibitor of apoptosis protein; ns, non-silencing; shRNA, short hairpin RNA. is a redox-regulated transcription factor that is activated during hypoxia (2Guo G. Bhat N.R. Antioxid. Redox. Signal. 2006; 8: 911-918Crossref PubMed Scopus (55) Google Scholar, 3Qanungo S. Wang M. Nieminen A.L. J. Biol. Chem. 2004; 279: 50455-50464Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). NFκB belongs to the Rel family, which includes five mammalian Rel/NFκB proteins: RelA (p65), c-Rel, RelB, NFκB1 (p50/p105), and NFκB2 (p52/p100) (4Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2470) Google Scholar). The inactive form of NFκB is localized in the cytoplasm as p65:p50 (the most abundant form) or p50:cRel heterodimers through interaction with IκB repressor proteins (IκBα,IκBβ,IκBγ, and IκBϵ) (5Kabe Y. Ando K. Hirao S. Yoshida M. Handa H. Antioxid. Redox. Signal. 2005; 7: 395-403Crossref PubMed Scopus (453) Google Scholar). Once activated, NFκB translocates to the nucleus, where it binds to DNA and activates various target genes including Bcl-xL, Bcl-2, a hematopoietic-specific Bcl-2 homologue A1, caspase-8-FADD-like interleukin-1β-converting enzyme inhibitory protein, tumor necrosis factor receptor-associated factors 1 and 2, cellular inhibitors of apoptosis, and X chromosome-linked inhibitor of apoptosis (XIAP/hILP) (6Braun T. Carvalho G. Fabre C. 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NFκB has cysteine residues close to or within its DNA-binding loops (9Ghosh G. van Duyne G. Ghosh S. Sigler P.B. Nature. 1995; 373: 303-310Crossref PubMed Scopus (511) Google Scholar), e.g. Cys59 of p50 and Cys38 of p65, that contact the DNA backbone (10Berkowitz B. Huang D.B. Chen-Park F.E. Sigler P.B. Ghosh G. J. Biol. Chem. 2002; 277: 24694-24700Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Cys62 of p50 is required for DNA binding activity (11Klatt P. Lamas S. Eur. J. Biochem. 2000; 267: 4928-4944Crossref PubMed Scopus (660) Google Scholar). Previously, changes in cellular redox state and disulfiram (thiocarbamate) treatment were shown to induce S-glutathionylation and subsequent inactivation of the p50 subunit of NFκB (11Klatt P. Lamas S. Eur. J. Biochem. 2000; 267: 4928-4944Crossref PubMed Scopus (660) Google Scholar, 12Brar S.S. Grigg C. Wilson K.S. Holder Jr., W.D. Dreau D. Austin C. Foster M. Ghio A.J. Whorton A.R. Stowell G.W. Whittall L.B. Whittle R.R. White D.P. Kennedy T.P. Mol. Cancer Ther. 2004; 3: 1049-1060PubMed Google Scholar). Because only p65-NFκB is responsible for transcriptional activation (13Huang D.B. Phelps C.B. Fusco A.J. Ghosh G. J. Mol. Biol. 2005; 346: 147-160Crossref PubMed Scopus (44) Google Scholar), it follows that glutathionylation of the transcriptionally active subunit p65 should have a more profound effect on NFκB activation than glutathionylation of the p50 subunit. The GSH/GSSG equilibrium is the major redox buffer in cells. Apart from providing cells with a reducing environment ([GSH] ≫ [GSSG]) and maintaining proteins in a reduced state, the glutathione redox couple dynamically regulates protein function via reversible disulfide bond formation (14Mieyal J.J. Gravina S.A. Mieyal P.A. Srinivasan U. Starke D.W. Packer L. Cadenas E. Biothiols in Health and Disease. Marcel Dekker, Inc., , New York1995: 305-372Google Scholar, 15Klatt P. Molina E.P. De Lacoba M.G. Padilla C.A. Martinez-Galesteo E. Barcena J.A. Lamas S. FASEB J. 1999; 13: 1481-1490Crossref PubMed Scopus (251) Google Scholar). Many modifications of protein sulfhydryls occur during oxidative stress, including formation of sulfenic, sulfinic, and sulfonic acids (16Biswas S. Chida A.S. Rahman I. Biochem. Pharmacol. 2006; 71: 551-564Crossref PubMed Scopus (464) Google Scholar). However, S-glutathionylation (i.e. formation of mixed disulfides between protein thiols and GSH (protein-SSG)) is likely the most prominent sulfhydryl modification (11Klatt P. Lamas S. Eur. J. Biochem. 2000; 267: 4928-4944Crossref PubMed Scopus (660) Google Scholar, 17Shelton M.D. Chock P.B. Mieyal J.J. Antioxid. Redox. Signal. 2005; 7: 348-366Crossref PubMed Scopus (327) Google Scholar). Moreover, reversible protein-SSG formation has also gained prominence as a mechanism of cellular regulation in redox-activated signal transduction (17Shelton M.D. Chock P.B. Mieyal J.J. Antioxid. Redox. Signal. 2005; 7: 348-366Crossref PubMed Scopus (327) Google Scholar). Consistent with a regulatory role, S-glutathionylation inactivates some proteins, e.g. glyceraldehyde-3-phosphate dehydrogenase, protein-tyrosine phosphatase (protein-tyrosine phosphatase 1B), and nuclear factor-1; whereas activating others like human immunodeficiency virus-1 protease, microsomal glutathione S-transferase, and hRas (17Shelton M.D. Chock P.B. Mieyal J.J. Antioxid. Redox. Signal. 2005; 7: 348-366Crossref PubMed Scopus (327) Google Scholar). An oxidative environment with high GSSG can promote protein-SSG formation. However, protein-SSG formation typically occurs intracellularly without substantial changes in the GSH/GSSG ratio, indicating alternative mechanisms of protein-SSG formation that are incompletely understood (18Ghezzi P. Biochem. Soc. Trans. 2005; 33: 1378-1381Crossref PubMed Scopus (109) Google Scholar, 19Gallogly M.M. Mieyal J.J. Curr. Opin. Pharmacol. 2007; (in press)PubMed Google Scholar). The enzyme glutaredoxin (GRx), also called thioltransferase, reverses protein glutathionylation in a specific and efficient manner. Accordingly, GRx is used to characterize oxidant-induced protein modification as protein-SSG (17Shelton M.D. Chock P.B. Mieyal J.J. Antioxid. Redox. Signal. 2005; 7: 348-366Crossref PubMed Scopus (327) Google Scholar). Although GRx is normally regarded as a deglutathionylating enzyme, GRx also catalyzes the reverse reaction, namely GSSG-dependent protein-SSG formation, and remarkably GRx can also enhance the rate of S-glutathionylation of proteins, e.g. actin, glyceraldehyde-3-phosphate dehydrogenase, and protein-tyrosine phosphatase 1B, by an alternative mechanism in the presence of glutathione thiol radical (GS.), despite high GSH content (20Starke D.W. Chock P.B. Mieyal J.J. J. Biol. Chem. 2003; 278: 14607-14613Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The p50 subunit of NFκB is reported to be inactivated by S-glutathionylation, but thus far no report has described an oxidative modification of the p65 subunit. Here we report that glutathione supplementation with N-acetyl-l-cysteine (NAC) decreases resistance to hypoxia in pancreatic cancer cells by inhibition of p65-NFκB binding to DNA. The inhibition of NFκB is reversible by GRx1 in vitro, and the inhibition is diminished in cells where GRx1 is knocked down, both characteristics indicative of S-glutathionylation of p65-NFκB as the mechanism of inhibition. Thus, S-glutathionylation and inactivation of NFκB leading to hypoxic cancer cell death may provide a useful adjunct to chemotherapy against hypoxic tumors. Chemicals and Reagents—Monoclonal anti-NFκB p65, anti-actin, anti-YY-1, and horseradish peroxidase-linked antibodies were obtained from Santa Cruz Biotechnology. Monoclonal antibody against poly(ADP-ribose) polymerase (PARP) was purchased from Trevigen (Gaithersburg, MD). Propidium iodide and Alexa 488-conjugated goat anti-mouse IgG were from Molecular Probes (Eugene, OR), DRAQ-5 from Alexis Biochemicals (San Diego, CA), and JSH-23 from Calbiochem (La Jolla, CA). The luciferase reporter construct pNFκB-Luc was from Stratagene (La Jolla, CA). d-NAC was a gift from Dr. Ian Cotgreave (AstraZeneca, Stockholm, Sweden). Recombinant GRx was isolated and purified as described previously (21Yang Y. Jao S. Nanduri S. Starke D.W. Mieyal J.J. Qin J. Biochemistry. 1998; 37: 17145-17156Crossref PubMed Scopus (131) Google Scholar). NADPH was from Roche Molecular Biochemicals. [35S]Glutathione was from ICN Radiochemicals (San Diego, CA). GSH, GSSG reductase (yeast), mono-carboxymethyl-BSA (BSA-CM), and all other chemicals were purchased from Sigma. Cell Culture—Human MIA PaCa-2 pancreatic carcinoma cells were obtained from the American Type Culture Collection (Rockville, MD). 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. Cells were grown to a 60-70% confluence before treatments. Generation of the GRx1 Knock-down Cell Line—Phoenix amphotropic cells were transfected with a puromycin-selectable pSUPER.retro.puro retroviral vector (OligoEngine, Seattle, WA) containing a specific short hairpin oligonucleotide sequence against GRx1 (GRx1-shRNA) or non-silencing sequence (ns-shRNA) using Lipofectamine PLUS (Invitrogen). Viral supernatants were collected and used to infect MIA PaCa-2 cells. Cells were selected and grown in puromycin (0.5 μg/ml) containing medium and screened for GRx1 protein expression and activity. Hypoxia Experiments—MIA PaCa-2 cells were cultured on 60-mm Petri dishes (5 × 105 cells/dish) for 42 h prior to exposure to hypoxia. Subsequently, cells were preincubated in the presence and absence of NAC for 3 h in complete culture medium supplemented with 10 mm glucose and 25 mm HEPES buffer. Petri dishes were placed in hypoxic Plexiglass chambers (Billups-Rottenberg, Del Mar, CA) and sparged with 95% N2, 5% CO2; 0.5% O2, 94.5% N2, 5% CO2; or 1% O2, 94% N2, 5% CO2 at 2 p.s.i. for 4 min. Chambers were kept at 37 °C throughout the course of the experiment. The oxygen concentration of the medium inside chambers was monitored continuously for 24 h using a FOXY-18G Fiber Optic Oxygen Sensor (Ocean Optics Inc., Dunedin, FL). After an initial equilibration of oxygen between gas phase and the medium, medium oxygen concentration inside the chamber remained constant during >24 h, indicating no oxygen back diffusion into the chambers. Nevertheless, some residual oxygen did remain inside the chambers (see "Results"). Transfection and Reporter Assay for NFκB Activity—MIA PaCa-2 cells (5 × 105 cells/dish) were plated on 60-mm Petri dishes for 30 h. Subsequently, cells were transiently transfected with 2 μg of plasmid containing 5× NFκB promoter fused to the firefly luciferase gene (Stratagene) or 2 μg of empty vector luciferase construct using Lipofectamine 2000 (Invitrogen). To normalize the transfection efficiency, cells were co-transfected with 2 μg of β-galactosidase control vector. Sixteen hours post-transfection, cells were pretreated for 3 h with NAC (10 mm), exposed to hypoxia or normoxia for 18 h, and then harvested. Relative luciferase activity was measured with a luciferase assay system (Promega, Madison, WI) using a luminometer (Turner Design, Sunnyvale, CA). 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. NFκB Activation and Binding—Specific binding of activated NFκB was measured with a Trans-AM™ NFκB kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. 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 the oligonucleotide. Epitopes on p50, p52, p65, c-Rel, and Rel-B are accessible only when NFκB is bound to its target DNA. Nuclear extracts (5-10 μg) were added to the wells followed by primary antibody against p65 and the horseradish peroxidase-conjugated secondary antibody. The optical density was measured at 450 nm with an absorbance plate reader. Preparation of BSA-[35S]SSG—BSA-[35S]SSG was prepared as described earlier with the following modifications (22Srinivasan U. Mieyal P.A. Mieyal J.J. Biochemistry. 1997; 36: 3199-3206Crossref PubMed Scopus (109) Google Scholar). After the reaction of S-carboxymethyl-BSA with N-succinimidylpyridyl bis(3,3′-dithiopropionate) and quenching with glycine, BSA was separated from small molecules either by gel filtration chromatography or by dialysis against two changes of 100 mm sodium phosphate buffer, pH 7.0. The modified BSA was then treated with excess [35S]GSH for 1 h at room temperature. The resulting BSA-[35S]SSG product was separated from [35S]GSH as described previously and typically had ∼0.9 GS eq/mol of BSA (22Srinivasan U. Mieyal P.A. Mieyal J.J. Biochemistry. 1997; 36: 3199-3206Crossref PubMed Scopus (109) Google Scholar). Radiolabel Assay for Glutaredoxin Activity—Whole cell lysates were analyzed for GRx activity with a radiolabel assay, which monitors the time-dependent release of radioactivity from BSA-[35S]SSG (23Gravina S.A. Mieyal J.J. Biochemistry. 1993; 32: 3368-3376Crossref PubMed Scopus (277) Google Scholar). Aliquots of lysate and assay buffer (0.1 m Na+/K+ phosphate buffer, pH 7.4, containing 0.5 mm GSH and 0.2 mm NADPH) were pre-warmed to 30 °C and mixed with an aliquot of BSA-[35S]SSG (final concentration of 0.1 mm) to initiate the reaction (total volume 0.5 ml). Aliquots of the reaction mixtures were precipitated with ice-cold trichloroacetic acid (final concentration of 10%) at 12 s, 1, 2, and 3 min, respectively. After centrifugation, the supernatants were analyzed for 35S by scintillation counting. Total rates of deglutathionylation (slopes of [35S]GSH released versus time) were corrected for non-enzymatic deglutathionylation by subtracting the rate of [35S]GSH released by GSH in the absence of cell lysate. Enzymatic rates were expressed as nanomole of product/min/mg of protein. Nuclear and Cytoplasmic Extracts—Nuclear and cytoplasmic extracts were prepared according to a modification of the method of Wang et al. (24Wang G.L. Jiang B.H. Semenza G.L. Biochem. Biophys. Res. Commun. 1995; 212: 550-556Crossref PubMed Scopus (159) Google Scholar). Briefly, cells cultured on 150-mm Petri dishes were scraped into 1 ml of ice-cold phosphate-buffered saline. Cells were centrifuged at 1,600 × g for 5 min at 4 °C. Cell pellets were lysed in 300 μl of low salt buffer (20 mm HEPES, 20% glycerol, 10 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, and 0.1% Triton X-100, pH 7.6) on ice. After a 20-min incubation, supernatants were collected as cytoplasmic extracts after centrifugation at 4,000 × g for 5 min at 4 °C. Nuclear pellets were then resuspended in high salt buffer (20 mm HEPES, 20% glycerol, 500 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, and 0.1% Triton X-100, pH 7.6) and tubes were rotated for 30 min at 4 °C, centrifuged at 16,000 × g, and supernatants were saved as nuclear extracts. In Vitro Deglutathionylation Assay—Recombinant GRx1 was isolated as described previously (25Chrestensen C.A. Starke D.W. Mieyal J.J. J. Biol. Chem. 2000; 275: 26556-26565Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Nuclear extracts were prepared as described above except 10 mm N-ethylmaleimide (NEM) was included in lysis buffer to block all free thiols. NEM-containing nuclear extracts were processed through spin columns (cut off 600 Da) to rid excess free NEM that potentially would inactivate the subsequent enzymatic reaction with GRx. Extracts were divided into different parts. One part was untreated and the other part was subdivided and aliquots were incubated with one of the following: (a) 4 mm dithiotheitol (DTT), (b) GRx1 plus GRx reducing system/recycling buffer (containing 0.2 mm NADPH, 0.5 mm GSH and 2 units/ml GSSG reductase), (c) recycling buffer alone. The assay was done with varying GRx1 concentrations using 1 μm (0.023 units), 100 nm, and 10 nm GRx1. The incubation times for DTT and GRx1 were 10 and 5 min, respectively. GRx1, along with its recycling system, efficiently converts glutathione-protein disulfides to free protein thiols and GSH. NEM binds irreversibly to free sulfhydryl groups but does not react with cysteine residues that are already oxidized. Subsequently, p65-NFκB DNA binding activity was measured by ELISA. 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, 50 mm Tris-Cl, pH 7.4) supplemented with a mixture of protease inhibitors (Roche Diagnostics). The lysates were centrifuged, and the resulting supernatants were assayed for total protein content (Bio-Rad). Equivalent amounts of protein were diluted in sample buffer (200 mm Tris-Cl, 15% glycerol, 10% SDS, 5% β-mercaptoethanol, 0.01% bromphenol blue, pH 6.8) and resolved on SDS-polyacrylamide gel. The proteins were then transferred and immobilized on 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—MIA PaCa-2 cells were cultured on 35-mm glass bottom Petri dishes (MatTek Corp., Ashland, MA). Cells were fixed with 1% paraformaldehyde for 30 min at room temperature and permeabilized with 0.25% Triton X-100 for 5 min at room temperature. After washes using Tris-buffered saline with 0.1% Tween 20 (TBST), cells were incubated with blocking solution (1% BSA in TBST) for 1 h followed by primary monoclonal antibody against p65 NFκB (1:100) overnight at 4 °C. Cells were then washed three times with TBST and incubated with anti-mouse secondary Alexa Fluor 488 (1:500) for 1 h at room temperature, followed by three washes with phosphate-buffered saline. DRAQ-5 (1 μm) was included in the final wash to stain the nuclei. Images of Alexa Fluor 488 fluorescence were collected using 488-nm excitation and >505-nm emission filters. DRAQ-5 fluorescence was imaged with a 633-nm excitation light and >640-nm emission filter using a Zeiss 510 NLO laser scanning confocal microscope. Caspase-3 Activation—Caspase-3 activity was measured using a Caspase-Glo™ 3/7 kit (Promega) according to the manufacturer's instructions. At an indicated time point, cultured MIA PaCa-2 cells were collected into a test tube followed by centrifugation. The pellet was resuspended and lysed with RIPA buffer. Caspase-Glo™ 3/7 reagent and the lysate were mixed in 1:1 ratio, and luminescence was measured with a luminometer. The resulting luminescence is proportional to caspase activity. Apoptosis Assay—Apoptotic cell death was determined from nuclear morphology after propidium iodide staining in the presence of digitonin. At the indicated time points, floating and adherent cells were collected, centrifuged, and resuspended in phosphate-buffered saline containing 200 μm digitonin (Calbiochem, La Jolla, CA) and 25 μm propidium iodide (Molecular Probes). Digitonin permeabilizes the plasma membrane and allows propidium iodide to enter cells and stain all nuclei. Thus, propidium iodide staining in the presence of digitonin is equivalent to staining with Hoechst and 4′,6-diamidino-2-phenylindole, the two commonly used fluorescent dyes in the literature to assess apoptosis on a cell by cell basis. Apoptotic nuclei were scored as apoptotic based on nuclear condensation and fragmentation and counted by a ×40 microscope objective using a rhodamine filter set and expressed as a percentage of total cells. At least 200 cells were counted from three different microscopic fields for each sample. In addition, we previously showed that apoptotic death after hypoxia plus NAC treatment assessed by propidium iodide and digitonin correlates closely with apoptotic death assessed with a Cell Death Detection ELISA PLUS kit (Roche Applied Science), which measures mono- and oligonucleosomes of 180-bp multiples formed in the cytoplasmic fractions during apoptosis (3Qanungo S. Wang M. Nieminen A.L. J. Biol. Chem. 2004; 279: 50455-50464Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Statistical Analysis—Data are presented as mean ± 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). A p value <0.05 was considered statistically significant. NAC-induced Apoptosis Is Oxygen-dependent in Pancreatic Cancer Cells—Our previous study showed that NAC, a GSH generating agent, enhanced hypoxic apoptosis in transformed murine embryonic fibroblasts (MEFs) by a mechanism apparently involving GSH-dependent suppression of NFκB transactivation (3Qanungo S. Wang M. Nieminen A.L. J. Biol. Chem. 2004; 279: 50455-50464Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Because several hypoxia-dependent genes are activated within a narrow O2 concentration range, we wanted to determine the window within which NAC is capable of enhancing hypoxic apoptosis in cancer cells. We exposed MIA PaCa-2 cells to different concentrations of O2 in the presence and absence of NAC (10 mm). Based on direct measurements with an oxygen probe, O2 concentrations in chambers flushed with 95% N2, 5%CO2; 0.5% O2, 94.5% N2, 5%CO2; and 1% O2, 94% N2, 5%CO2 were 0.6, 1.6, and 2.1% O2, respectively. Thus, even in chambers with 95% N2, 5%CO2, medium O2 content did not reach zero (anoxia), possibly due to residual O2 in the gas mixture and outgassing of O2 from plastic Petri dishes and other plastic parts inside the chamber. Hypoxia alone for 22 h in 2.1, 1.6, and 0.6% O2 induced very little apoptosis (1.9% during normoxia versus 4.7, 3.7, and 3.1%, respectively). In contrast, NAC (10 mm) enhanced hypoxic apoptosis in a O2-dependent manner (2.4% during normoxia versus 10.3, 19.6, and 22.6%, respectively) (Fig. 1A). NAC is rapidly taken up by the cells. Once inside the cells, endogenous esterases release free l-cysteine, which is a precursor of GSH synthesis (26Jiang B. Brecher P. Hypertension. 2000; 35: 914-918Crossref PubMed Scopus (33) Google Scholar). Like NAC, GSH ester (5 mm) enhanced apoptosis from 2.4% (normoxia) to 24% after 22 h exposure to hypoxia (1.6% O2) (Fig. 1B). To confirm that NAC-mediated enhancement of apoptosis in hypoxic cells was due to an increase in GSH, we incubated cells with the d-isomer of NAC (d-NAC), which is not a precursor of GSH synthesis. Unlike the l-isomer of NAC, the d-isomer did not promote hypoxic apoptosis in MIA PaCa-2 cells (Fig. 1B). NAC activated caspase-3 in hypoxic cells indicative of apoptotic death (Fig. 1C). PARP is a target of cysteine proteases, and caspase-3 cleaves 116-kDa PARP into a 85-kDa fragment during apoptosis (27Qanungo S. Basu A. Das M. Haldar S. Oncogene. 2002; 21: 4149-4157Crossref PubMed Scopus (58) Google Scholar). Hypoxia alone did not cause PARP cleavage assessed by Western blot analysis. In contrast, hypoxia in combination with NAC (10 mm) produced the 85-kDa cleavage product of PARP (Fig. 1D). Collectively, the results from Fig. 1 indicate that elevated GSH is responsible for enhancing hypoxic apoptosis in pancreatic cancer cells in agreement with our previous results in MEFs (3Qanungo S. Wang M. Nieminen A.L. J. Biol. Chem. 2004; 279: 50455-50464Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Inhibition of NFκB Activation Is Oxygen-dependent in MIA PaCa-2 Cells—Hypoxia alone (1.6 and 0.6% O2) increased NFκB luciferase activity by 2.4- and 2.8-fold, respectively, compared with normoxia. The increase was blocked by NAC (Fig. 2A). To determine whether blockade of hypoxia-induced transcriptional activation of NFκB was due to decreased p65-NFκB binding to DNA, NFκB DNA binding was measured. Hypoxia (1.6% O2) alone (18 h) increased p65-NFκB DNA binding 2.1-fold (compared with normoxia), which was abolished by NAC (Fig. 2B). NAC also affected NFκB target genes. In our previous study, we identified XIAP as a target gene for NFκB during hypoxia to MEFs (3Qanungo S. Wang M. Nieminen A.L. J. Biol. Chem. 2004; 279: 50455-50464Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). We also confirmed NFκB-dependent activation of XIAP in MIA PaCa-2 cells during hypoxia, which was prevented by NAC (data not shown). The results indicated that NAC treatment inhibited transcriptional activation of NFκB thereby inhibiting the survival of hypoxic cancer cells. NAC Does Not Change NFκB Protein Levels—NFκB inactivation by NAC may also b
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