| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 279, Issue 48, 50455-50464, November 26, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
B in Hypoxic Murine Embryonic Fibroblasts*
From the Department of Anatomy and Case Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106
Received for publication, June 16, 2004 , and in revised form, August 19, 2004.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B (NFB) 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 B(IB
), NFB-p65 translocation into the nucleus, NFB binding to DNA, and subsequent transactivation of NFB, which increased X chromosome-linked inhibitor of apoptosis protein levels. NAC failed to block degradation by IB and sequestration of the p65 subunit of NFB to the nucleus. However, NAC did abrogate hypoxia-induced NFB binding to DNA, NFB-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 NFB transactivation. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B (NFB).1 NFB is a critical transcription factor that tips the balance from apoptosis of cells to proliferation and malignant growth of tumor cells. NFB belongs to the Re1 family, which includes five mammalian Re1/NFB proteins: Re1A (p65), c-Re1, Re1B, NFB1 (p50/p105), and NFB2 (p52/p100) (4). The activity of NFB is regulated by movement between the cytoplasm and nucleus in response to cell stimulation (5). NFB dimers containing Re1A or c-Re1 are retained in the cytoplasm through interaction with inhibitor of B(IB) repressor proteins (IB, IB
, IB
, and IB
) (6). Potent NFB activators, such as tumor necrosis factor-, interleukin-1, and lipopolysaccharide, cause IB kinase (IKK)-mediated phosphorylation of IB on serine 32 and serine 36 residues, followed by ubiquitination at the nearby lysine residues and proteasomal degradation (7). This modification of IB exposes the nuclear localization signal of NFB and leads to NFB translocation to the nucleus, presumably as a consequence of binding to karyopherins (8), proteins responsible for nucleocytoplasmic transport in cells.
An alternative, less-characterized pathway of NFB activation is by tyrosine 42 phosphorylation of IB after hypoxia/reoxygenation or pervanadate treatment (6). However, in T-cells and bone marrow macrophages, tyrosine phosphorylation of IB is insufficient to activate NFB, suggesting that tyrosine and serine kinases act at multiple levels to dissociate the IB/NFB complex (8). Once IB is inactivated, NFB dimers are free to localize to the nucleus, where they undergo further modification, mostly through phosphorylation of the Re1 proteins (9). Activated NFB 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 (7, 10). NFB suppresses apoptosis by inducing expression of various anti-apoptotic genes, including Bcl-xL, Bcl-2, hematopoietic-specific Bcl-2 homolog A1 (Bfl1) (11), caspase-8-FADD-like interleukin-1-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) (7). NFB also regulates X chromosome-linked IAP (XIAP/hILP), which inhibits caspase-3 and caspase-7 and prevents activation of pro-caspase-9 (12).
Hypoxia promotes reactive oxygen species (ROS) formation in a number of cell types (13–15). ROS, such as H2O2, may act as second messengers activating NFB (16). Hyperoxia, which also induces ROS generation, causes nuclear translocation and activation of NFB (17). Conversely, increased cellular antioxidants prevent NFB activation by cytokines (18). Although substantial evidence supports a role of ROS in NFB activation, the contribution of redox regulation to NFB activation is still controversial (19).
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-1-induced inducible nitric oxide synthase expression through an oxidation/reduction-related mechanism potentiating cytokine activation of the p44/42 MAPK signaling cascade (20, 21). NAC inhibits activation of c-Jun N-terminal kinase, p38 MAPK, redox-sensitive activator protein-1, and NFB transcription factor activities regulating the expression of numerous genes (21, 22). Although several reports show that NAC protects against oxidative stress-induced cell death (23, 24), 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 (22).
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, NFB activation, and cell survival in hypoxic transformed cells. We show that (i) NAC potentiates apoptosis of hypoxic transformed cells, and (ii) NAC inhibits NFB-dependent expression of antiapoptotic proteins such as XIAP.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B p65, polyclonal anti-IB, 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, F1-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. -Methylomuralide was obtained from Calbiochem (La Jolla, CA). The luciferase reporter construct pNFB-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 x 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 x 106 cells/dish) were plated on 60-mm Petri dishes for 24 h. Subsequently, cells were transiently transfected with 1 µg of plasmid containing 2x NFB promoter fused to the firefly luciferase gene (10) 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 -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 -galactosidase activity, which was measured using a FluoReporter LacZ/-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 NFB.
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) (25). 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 x 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 (26).
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% -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 NFB (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. (27). 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 mM N-(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 x 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 x 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 x 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 (28).
NFB Activation—NFB activation was measured with a Trans-AM NFB kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. In this assay, the oligonucleotide containing the NFB consensus sequence (5'-GGGACTTTCC-3') is immobilized onto a 96-well plate. Only the active form of NFB in the cell extract specifically binds to this oligonucleotide. Epitopes on p50, p52, p65, c-Re1, and Re1B are accessible only when NFB 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 40x 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 (29). 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.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B activation in cancer cells (19, 30).
|
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.
|
NAC Blocks Hypoxia-induced ROS Formation—NAC is usually associated with protection against ROS-dependent cell killing (32). Here, however, NAC enhanced hypoxia-induced apoptosis (Fig. 1). Accordingly, we assessed the effect of NAC on ROS generation during hypoxia. ROS were detected with CM-H2DCFDA, which becomes highly fluorescent DCF when oxidized by ROS (13). Exposure of MEFs to 4 h of hypoxia increased DCF fluorescence by 65% compared with normoxia (Fig. 3A). NAC almost completely abolished hypoxia-induced DCF fluorescence, indicating that NAC blocked hypoxia-induced ROS formation. We also tested the effect of other antioxidants on hypoxia-induced ROS generation. Trolox, a vitamin E analog (1 mM), and polyphenolic antioxidant epigallocatechin-3-gallate (EGCG) (100 µM) also prevented ROS formation during hypoxia (Fig. 3A). Trolox was even better at scavenging ROS than NAC. However, unlike NAC, neither trolox nor EGCG enhanced hypoxia-induced apoptosis (Fig. 3B). The results indicate that NAC-mediated enhancement of apoptosis was not a direct consequence of inhibition of ROS formation.
|
Glutathione Is Involved in Hypoxia-induced Apoptosis by NAC—NAC is rapidly taken up by cells. Inside cells, endogenous esterases release free L-cysteine, which is a precursor for GSH synthesis (20). To confirm that NAC is capable of increasing intracellular GSH levels, we measured intracellular GSH with high-performance liquid chromatography. NAC increased GSH in normoxic and hypoxic cells (Fig. 4A). After 4 h of exposure to hypoxia, NAC-treated cells had higher GSH compared with untreated cells (Fig. 4A).
|
Cellular acidosis is usually associated with hypoxia in solid tumors. To exclude the possibility that a decrease of pH was responsible for enhanced apoptosis induced by NAC and GSH monoester, we measured the pH of the medium in the presence and absence of NAC during normoxia and hypoxia. Exposure to 12 h of hypoxia decreased medium pH by 0.1 pH unit compared with normoxia. NAC and D-NAC further decreased the medium pH by 0.3 units compared with hypoxia alone. However, only NAC promoted hypoxia-induced apoptosis (Fig. 4C). GSH monoester did not change pH during hypoxia but did promote apoptosis during hypoxia (Fig. 4B). Therefore, we conclude that enhanced hypoxic apoptosis by NAC and GSH monoester was not attributable to decreased pH but rather attributable to increased GSH. These results are in agreement with the recent study with human RKO colorectal carcinoma cells, where changes in medium pH between 7.3 and 6.9 had no effect on apoptosis (30).
NAC Does Not Prevent Hypoxia-induced IB Degradation and NFB Translocation to the Nucleus—Because NAC did not block activation of the MAPK survival pathway (Fig. 3C), we turned our attention to the redox-sensitive transcription factor NFB, which is activated by numerous factors, including ROS (8). NFB activation is negatively regulated by a family of IB repressor proteins, of which the most extensively studied is IB. Ubiquitin-mediated proteolytic degradation of IB leads to the translocation of NFB from the cytoplasm to the nucleus, where it activates transcription. To determine the role, if any, of NFB signaling in NAC-mediated promotion of apoptosis, we determined the effect of NAC on IB protein levels. MEFs were exposed to hypoxia for 4 h in the presence and absence of NAC. Cytosolic fractions were prepared, and IB proteins were analyzed with Western blotting. As expected, hypoxia induced IB protein degradation (Fig. 5A). Surprisingly, NAC failed to prevent IB protein degradation. To confirm that IB protein degradation after hypoxia was mediated by proteasomes, we measured IB protein levels with and without 20 µM -methylomuralide, a cell-permeable, irreversible inhibitor of proteasome function (34). Methylomuralide equally inhibited IB degradation during hypoxia and hypoxia plus NAC (Fig. 5A).
|
B was also performed after 4 h hypoxia in the presence and absence of NAC. During normoxia, p65 was primarily localized to the cytoplasm, and large dark voids in the confocal image corresponded to the Hoechst-stained nuclei (Fig. 5B). These findings indicated that NFB was localized to the cytoplasm and therefore was not activated. During hypoxia, p65 became localized to the nuclei, showing directly NFB translocation to the nucleus (Fig. 5B). However, NAC treatment did not prevent p65-NFB translocation to the nucleus during hypoxia (Fig. 5B). These results demonstrate that NAC does not affect proteasomal degradation of IB or NFB nuclear translocation during hypoxia.
NAC Inhibits Hypoxia-induced Transactivation of NFB— Although hypoxia induced NFB nuclear translocation, transcriptional activation of nuclear NFB may be blocked by other factors. Therefore, a gene reporter assay for NFB was performed. Hypoxia increased NFB luciferase reporter activity by 150% compared with normoxic activity (Fig. 6A). NAC prevented the increase of reporter activity completely (Fig. 6A). We next determined whether increased transcriptional activation of NFB was attributable to increased p65-NFB binding to DNA, as assessed by a p65-NFB-specific ELISA using nuclear extracts (Fig. 6B). In this approach, the NFB response element DNA was attached to microtiter plate wells, and nuclear NFB capable of binding this sequence was measured by ELISA. Hypoxia alone (4 h) increased p65-NFB DNA binding compared with normoxia. NAC decreased hypoxia-induced p65-NFB DNA binding (Fig. 6B). By contrast, the non-thiol antioxidants trolox and EGCG did not reverse hypoxia-induced increase of p65-NFB DNA binding (data not shown). Taken together, these results indicate that hypoxia activated NFB translocation to the nucleus and enhanced NFB binding to DNA, resulting in activation of NFB-dependent gene expression. NAC treatment during hypoxia did not abolish NFB nuclear translocation but blocked DNA binding and subsequent gene activation.
|
B transcriptionally activates several anti-apoptotic target genes including Bcl-xL, Bcl-2, c-FLIP, XIAP, and cellular inhibitors of apoptosis (c-IAPs) (7). XIAP is of particular interest, because XIAP blocks apoptosis at a post-mitochondrial level by inhibiting caspase-9. Exposure of MEFs to 4 h of hypoxia increased protein expression of XIAP, an effect down-regulated by NAC (Fig. 7A). XIAP contains a caspase-binding domain and becomes bound to activated caspase-9, thus blocking caspase-3 activation and apoptosis (35). Exposure of oncogenic MEFs to 7 h of hypoxia did not increase caspase-3 activity compared with normoxia, suggesting that the hypoxia-driven increase in XIAP protein expression may interfere with caspase-3 activation (Fig. 7C). Hypoxia in the presence of NAC, by contrast, led to a 467% increase in caspase-3 activity (Fig. 7C), which paralleled apoptotic cell death (Fig. 1B). The effect of hypoxia on other anti-apoptotic genes that are transcriptionally activated by NFB was also determined. However, no change in the protein expression of Bcl-xL, Bcl-2, or c-FLIP was observed during hypoxia (Fig. 7B). Also, hypoxia did not alter the protein status of Bax (Fig. 7B). Taken together, we identified XIAP as a target gene for NFB activation during hypoxia.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
B signaling pathway. NAC did not inhibit NFB translocation to the nucleus (Fig. 5). However, NAC interfered with NFB-DNA binding and blocked hypoxia-induced NFB transcriptional activation (Fig. 6). As a consequence, a NFB-dependent XIAP expression was suppressed, which accounted, at least in part, for increased caspase-3 activation and enhanced apoptosis during hypoxia (Fig. 7).
Cellular sensors for hypoxia include hypoxia inducible factor-1, activator protein-1, and vascular endothelial growth factor, which are activated in response to hypoxia to allow cells to survive a hypoxic environment (36). Therefore, cancer cells are usually resistant against hypoxia (30, 37), in agreement with the results shown in this study (Fig. 1). Because ROS are formed during hypoxia (Fig. 2A), ROS may be required for activation of these survival genes (21). Hypoxia (1 to 5% O2) increases mitochondrial ROS production (14), and mitochondrial ROS may initiate signaling cascades such as NFB and MAPK to protect from cell death (38, 39). By contrast, decreases in intracellular ROS formation during hypoxia may allow expression of genes that are otherwise suppressed by ROS (40). Here we show that NAC increases hypoxia-induced apoptosis (Fig. 1). Our results agree with earlier reports that antioxidants such as NAC increase UV radiation-induced apoptosis in C8161 melanoma cells (41). However, NAC-mediated enhancement of apoptosis in hypoxic MEFs was not attributable to inhibition of p42/44 MAPK phosphorylation, as was the case for C8161 melanoma cells after UV radiation (41). Although hypoxia increased MAPK phosphorylation, a measure of MAPK activation, NAC did not prevent this phosphorylation (Fig. 3C).
To determine whether NAC-mediated enhancement of apoptosis was attributable to inhibition of ROS, we examined the effects of the antioxidants trolox and EGCG on ROS and cell viability. Similar to NAC, trolox and EGCG blocked hypoxia-induced ROS formation (Fig. 3A). Surprisingly, neither trolox nor EGCG enhanced hypoxic apoptosis (Fig. 3B). Therefore, NAC-mediated enhancement of hypoxic apoptosis did not appear to be the consequence of inhibition of ROS formation. Trolox is a vitamin E analog, whereas EGCG is a polyphenolic antioxidant. Neither is a precursor of GSH. Therefore, we hypothesized that NAC-mediated enhancement of apoptosis occurred via increased intracellular GSH. Accordingly, we measured intracellular GSH levels after treatment with NAC and GSH monoester, which also increases GSH. Both NAC and GSH monoester increased GSH during normoxia and hypoxia. After 4 h of treatment, GSH monoester increased intracellular GSH levels more than NAC (Fig. 4A). The differences between NAC and GSH monoester may relate to the time course of GSH supplementation by the two agents. GSH monoester loads GSH into cells more rapidly than NAC, as measured after 4 h (Fig. 4A), whereas NAC as a GSH precursor likely sustains high GSH levels over a longer time, up to 12 h when apoptosis was assessed. Thus, although both NAC and GSH monoester enhanced hypoxic apoptosis, GSH monoester appeared to promote hypoxic apoptosis somewhat less than NAC (Fig. 4B), because increased GSH levels after GSH monoester treatment likely declined more rapidly than after NAC treatment.
Further evidence that enhanced apoptosis is a consequence of increased GSH comes from the data that neither D-NAC nor DTT enhanced hypoxic apoptosis (Fig. 4C). D-NAC loads cells with D-cysteine and increases total thiol content, but D-cysteine cannot be used for GSH synthesis (42). Similarly, DTT promotes thiol reduction but does not increase GSH. Taken together, the present data indicate that NAC-mediated enhancement of hypoxic apoptosis was not a direct consequence of ROS inhibition but rather depended on GSH status.
ROS can cause direct cellular damage and can also act as second messengers for signal transduction pathways. In particular, NFB is a redox-regulated transcription factor that is important in signaling and in the development and progression of malignancies (7). The involvement of ROS in the activation of NFB is based on the observations that agents that stimulate ROS production activate NFB, whereas antioxidant compounds inhibit NFB activation (43–45). Another study suggests that ROS do not directly mediate NFB activation. The study proposes that NAC blocks tumor necrosis factor--stimulated NFB activation by inhibiting the binding of tumor necrosis factor- to its receptors without affecting interleukin-1- or 12-O-tetradecanoylphorbol-13-acetate-stimulated NFB activation, and that endogenous ROS from the Rac/NADPH oxidase system do not mediate NFB activation (19). Accordingly, we investigated whether NAC-mediated enhancement of hypoxic apoptosis was attributable to inhibition of NFB.
The activation of NFB is controlled by a family of IB repressor proteins (11). A critical step in NFB activation is IB phosphorylation by kinases such as IKK, which directs IB into 26 S proteasomes for degradation. Exposure to hypoxia for 4 h induced IB degradation, a step toward NFB activation (Fig. 5A). Although NAC blocked ROS formation, NAC did not prevent IB degradation. This finding indicated that ROS were not essential for IB inactivation and that NAC was not acting at this step in the NFB cascade.
Proteasome inhibition blocked IB degradation during hypoxia (Fig. 5A). Ubiquitin-conjugating enzymes are inhibited by high levels of oxidized GSH (45). In airway epithelial cells exposed to H2O2, inhibition of ubiquitin-dependent proteolysis results in sustained IB-dependent suppression of NFB and increased apoptosis (46). However, IB degradation during hypoxia to MEFs in the present work was not inhibited by NAC (Fig. 5A), which implies that increased GSH after NAC was not affecting the efficiency of IB proteolysis. In some systems, tyrosine 42 phosphorylation of IB suppresses IB-dependent NFB inhibition, an effect not mediated by proteasomal degradation of IB (6). This was not the case for MEFs during hypoxia, because IB was degraded both in the presence and absence of NAC (Fig. 5A). Another mechanism of IB inactivation is glutathione-dependent protein S-thiolation (47) of cysteine 179 in IKK and IKK (39). However, such mechanism does not account for NAC-dependent NFB inactivation during hypoxia, because the redox-sensitive site in the present study was downstream of IB degradation and subsequent p65 nuclear translocation (Fig. 5B).
During hypoxia, NFB translocated to the nucleus. NAC did not prevent this translocation (Fig. 5B). In the nucleus, NFB binding to promoter regions of NFB target genes depends on conserved cysteines. The redox state of these cysteines is maintained by the thioredoxin/thioredoxin reductase system (48). However, the GSH precursor NAC decreased rather than increased DNA binding by NFB (Fig. 6). These considerations argue against involvement of thioredoxin in hypoxia-induced NFB activation, but further studies will be needed to identify the molecular target(s) of NAC-mediated blockade of NFB activation during hypoxia.
Hypoxia increased XIAP protein expression, which was down-regulated by NAC (Fig. 7A). The hypoxia-dependent upregulation was specific for XIAP, because the other NFB target genes, including Bcl-xL, Bcl-2, Bax, and c-FLIP, remained unchanged during hypoxia (Fig. 7B). A similar effect of hypoxia on Bcl-xL, Bcl-2, and, Bax has been reported earlier in malignant glioma cells (49). XIAP suppresses apoptosis post-mitochondrially by inhibiting cytochrome c-dependent activation of caspase-9 and caspase-3. NAC, by decreasing XIAP expression, thus may enhance caspase activation after cytochrome c release. Consistent with this view is that NAC did not alter cytochrome c release during hypoxia (Fig. 2A) but did increase caspase-3 activation (Fig. 7C).
p53-deficient MEFs were virtually completely resistant to apoptosis after hypoxia and hypoxia plus NAC (data not shown), whereas hypoxia induced cytochrome c release and apoptosis in p53+/+ MEFs, especially in the presence of NAC (Fig. 2A). Hypoxia also increased p53 protein expression in p53+/+ MEFs.2 Thus, hypoxia activated a p53-dependent intrinsic pathway of apoptosis. The initial activation of this intrinsic pathway was not sensitive to NAC, because NAC did not alter cytochrome c release during hypoxia. Possible mechanisms of p53-dependent induction of the intrinsic apoptotic pathway involve increased expression of pro-apoptotic genes of the Bcl-2 family, such as Bax, Noxa, and PUMA (50–52). p53 can also directly activate Bax and trigger cytochrome c release (50). Although we did not observe an increase of total Bax protein during hypoxia (Fig. 7B), p53 protein may activate Bax translocation to mitochondria and therefore cause cytochrome c release in a p53-dependent manner (50). Two other pro-apoptotic proteins, PUMA and Noxa, are also induced in response to p53 and subsequently translocate to mitochondria to trigger cytochrome c release (51, 52). Further studies are required to define the roles these proteins play in p53-dependent cytochrome c release during hypoxic apoptosis.
In conclusion, activation of NFB-dependent survival pathways confers death resistance during hypoxia, a common feature of most solid tumors. Intracellular GSH supplementation with agents such as NAC and GSH monoester block this activation by preventing NFB from binding to and activating the promoter regions of its target genes. Thus, manipulations to increase cellular GSH levels could potentially be used therapeutically to sensitize cancer cells to hypoxic apoptosis. Down-regulation of XIAP, a NFB target gene, might also complement traditional anticancer therapy by selectively enhancing hypoxic apoptosis at a post-mitochondrial level (Fig. 8).
|
|
FOOTNOTES |
|---|
To whom correspondence should be addressed: Case Comprehensive Cancer Center Laboratories, Case Western Reserve University, 3-134 Wolstein Research Bldg., 10900 Euclid Ave., Cleveland, OH 44106-7285. Tel.: 216-368-5680; Fax: 216-368-8919; E-mail: axn25{at}case.edu.
1 The abbreviations used are: NFB, nuclear factor-B; IB, inhibitor of nuclear factor B; IKK, IB kinase; c-FLIP, caspase-8-FADD-like IL-1-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.
2 S. Qanungo, M. Wang, and A.-L. Nieminen, unpublished results.
|
ACKNOWLEDGMENTS |
|---|
B-luc plasmid, Dr. Ian Cotgreave for D-NAC, and Dr. Scott Lowe for the MEF cell line. We also thank Dr. John Lemasters for critical reading of the manuscript and helpful discussion. |
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, normoxia plus NAC; 
, hypoxia; 
, hypoxia plus NAC. C, MEFs were pre-incubated with 20 mM NAC as indicated and exposed to hypoxia for 12 h. Cells were harvested, and oligonucleosomal DNA fragments were measured in cytoplasmic fractions using a Cell Death ELISA DetectionPLUS kit. Absorbance of each sample was divided by absorbance of normoxic samples. #, p < 0.01 compared with hypoxia. D, MEFs were pre-incubated with 20 mM NAC as indicated and exposed to hypoxia for 12 h. Cells were scored for necrosis using trypan blue as described in "Materials and Methods." E, MIA PaCa-2 cells were pre-incubated with 20 mM NAC as indicated and exposed to hypoxia for 15 h. Cells were scored for apoptosis as in A. f, p < 0.0001 compared with hypoxia. F, A549 cells were pre-incubated with 20 mM NAC as indicated and exposed to hypoxia for 26 h. Cells were scored for apoptosis as in A. f, p < 0.0001 compared with hypoxia.
, p < 0.05 compared with normoxia; #, p < 0.01; *, p < 0.001 compared with hypoxia. B, apoptosis was assessed under the same experimental conditions as in A after 12 h of hypoxia using propidium iodide and digitonin. *, p < 0.001 compared with hypoxia. C, MEFs were treated with 20 mM NAC and 50 µM PD98059 for 1 h, followed by normoxia or hypoxia for 4 h as indicated. Cell lysates were subjected to Western blotting analysis. Numbers above the phopho-p44/42 bands indicate the band intensities normalized against normoxia.
