Nitric Oxide Disrupts H2O2-dependent Activation of Nuclear Factor kappa B

ROLE IN SENSITIZATION OF HUMAN TUMOR CELLS TO TUMOR NECROSIS FACTOR-alpha -INDUCED CYTOTOXICITY*

Hermes J. Garbán and Benjamin BonavidaDagger

From the Department of Microbiology, Immunology and Molecular Genetics, and Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, California 90095-1747

Received for publication, September 15, 2000, and in revised form, November 26, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tumor necrosis factor alpha  (TNF-alpha ) exerts its effect by two distinct signaling pathways. It can trigger cytotoxicity in sensitive target cells. TNF-alpha can also promote nuclear factor kappa B (NF-kappa B) activity and regulate the expression of genes that interfere with apoptosis and thus conferring resistance to several apoptotic stimuli. We have observed that interferon-gamma (IFN-gamma ) sensitizes human ovarian carcinoma cell lines to TNF-alpha -mediated apoptosis and further, IFN-gamma induces the expression of the inducible nitric-oxide synthase (iNOS) and the generation of nitric oxide (NO). This study examines the role of NO in the sensitization of the ovarian carcinoma cell line AD10 to TNF-alpha -mediated cytotoxicity. Treatment of AD10 cells with the NOS inhibitor L-NMA blocked the IFN-gamma -dependent sensitization whereas NO donors (S-nitroso-N-acetylpenicillamine) sensitized these cells to TNF-alpha cytotoxicity. Analysis of the activation status of NF-kappa B upon treatment with NO donors confirmed the inhibitory role of NO on both the NF-kappa B DNA-binding property and its activation. Moreover, the inhibition of NF-kappa B nuclear translocation by NO donors directly correlated with the intracellular concentration of H2O2 and was reversed by the addition of exogenous H2O2. These findings show that NO might interfere with TNF-alpha -dependent NF-kappa B activation by interacting with O&cjs1138;2 and reducing the generation of H2O2, a potent NF-kappa B activator. Therefore, NO-mediated disruption of NF-kappa B activation results in the removal of anti-apoptotic/resistance signals and sensitizes tumor cells to cytotoxic cytokines like TNF-alpha .


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The development of resistance to either the immune system or chemo-immunotherapeutic strategies remains a disadvantage in the therapy of cancer, particularly in cases where recurrences and/or relapses occurred. Apoptosis has been accepted as a distinct pathological mechanism in tumors responding to anticancer therapies. Further, resistance to apoptosis in tumor cells has been recognized as a common pathway to multiple drug resistance (1, 2). Multiple lines of evidence have implicated the activation of the transcription factor NF-kappa B1 as one of the primary signals in the onset of resistance to many apoptotic stimuli, particularly TNF-alpha (3-5).

TNF-alpha is a proinflammatory cytokine that exerts a broad spectrum of biological effects by its interaction with two distinct cell surface receptors, TNFR1 and TNFR2 (6). Most cytotoxic effects of TNF-alpha are mediated by the TNFR1. It has been demonstrated that, upon interaction with TNF-alpha , trimerization of TNFR1 takes place and results in cellular signaling leading to the recruitment of the TNFR1-associated death domain protein and the receptor-interacting protein to the receptor complex (7). The TNFR1-associated death domain protein interacts with the Fas-associated death domain to initiate the death pathway and engages several proteins such as the TNFR-associated factor-1, the TNFR-associated factor-2, and receptor-interacting protein to initiate the TNF signaling pathways such as the activation of NF-kappa B (8).

Reactive oxygen species (ROS) have also been implicated in the signaling pathways initiated by TNF-alpha . Stimulation of mammalian cells with TNF-alpha triggers the generation of various ROS (9, 10). Hence, the use of antioxidants results in the inhibition of various TNF-alpha -related effects such as the activation of transcription factors, gene expression, and cytotoxicity. In addition, the use of exogenous ROS mimics the biological activity of TNF-alpha (11). These data support the hypothesis that ROS function as second messengers for TNF-alpha -mediated signaling. In biological systems the most important ROS generated upon TNF-alpha stimulation are the result of enzymatic partial reduction of oxygen yielding superoxide (O&cjs1138;2), which is either immediately reduced by superoxide dismutase to hydrogen peroxide (H2O2) or alternatively reacts rapidly with nitric oxide (NO) to generate ONOO- (12-14). However, the regulatory role of NO in TNF-alpha signaling via the disruption of ROS-dependent activation of NF-kappa B has not been established.

Several lines of evidence showed that resistant tumors could be sensitized to TNF-alpha -mediated cytotoxicity by various cytokines or pharmacological treatments (15-20). Recently, we have reported that IFN-gamma induced the sensitization of the human ovarian carcinoma AD10 cell line to Fas-mediated apo ptosis and the sensitization was due in part to the generation of nitric oxide by the induction of iNOS in these cells (21). NO has been identified as a potential second messenger based on its ability to chemically interact with a broad range of regulatory proteins. Furthermore, NO can interact with metal cluster- and thiol-containing proteins (for review, see Ref. 22) resulting in the modification of both the structures and functions of these proteins. Although NO has been shown to react very rapidly with O&cjs1138;2, the only biological effect to this chemical reaction has been assigned to the generation of ONOO-, a proposed cytotoxic derivative (23, 24).

Herein, we hypothesize that NO is interfering with the TNF-alpha -mediated signaling by chemically reacting with O&cjs1138;2. Since can serve as a precursor to H2O2, which is a proposed activator of the anti-apoptotic transcription factor NF-kappa B, the reaction of O&cjs1138;2 with NO will interfere with the activation of NF-kappa B and will result in the removal of anti-apoptotic signals and sensitization of the tumor cells to TNF-alpha cytotoxicity. This study has been designed to test this hypothesis, and the following have been examined: (a) the molecular mechanism by which IFN-gamma sensitizes the human ovarian carcinoma cell line to TNF-alpha -induced cytotoxicity, (b) the specific role of NO in the disruption of TNF-alpha -mediated generation of H2O2, and, subsequently, (c) the mechanism by which NO can disrupt the TNF-alpha -dependent NF-kappa B activation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Reagents-- The AD10 cell line is an adriamycin-resistant, MDR phenotype-expressing subline derived from the human ovarian carcinoma cell line A2780 and was obtained from Dr. Ozols (Fox Chase Cancer Center, Philadelphia, PA). The PC-3 cell line is a metastatic bone-derived human prostatic adenocarcinoma, CRL-1435, obtained from ATCC (American Type Culture Collection, Manassas, VA). Cell cultures were maintained as monolayers on plastic dishes in RPMI 1640 medium (MediaTech, Inc., Herndon, VA), supplemented with 10% heat-inactivated FBS (Gemini Bio-Products, Inc., Calabasas, CA), 1% L-glutamine (Life Technologies, Inc.), 1% pyruvate (Life Technologies, Inc.), 1% nonessential amino acids (Life Technologies, Inc.), and incubated at 37 °C and 5% CO2. For every experimental condition, the cells were cultured in 1% FBS 24 h prior to treatments. In cases where SNAP (kindly provided and synthesized by Dr. Jon Fukuto, UCLA, Los Angeles, CA) was used, 500 µM photo-activated SNAP was added to the cultured cells 2 h prior to stimulation with cytokines unless otherwise indicated in the text. For iNOS induction, cultured cells were stimulated 18 h with 100 units of human recombinant IFN-gamma (PeproTech, Inc., Rocky Hill, NJ). For guanylate cyclase-related effects, cells were incubated in the presence of the cGMP analogue 8-bromo-cGMP instead of SNAP or blocked using 300 µM 1H-(1,2,4)oxadiazolo-[4,3-a]quinoxalin-1-one (Alexis Corp., San Diego, CA).

Cytotoxicity Assay-- TNF-alpha -mediated cytotoxicity was assessed using recombinant TNF-alpha at the concentrations of 0.01, 0.1, and 1 ng/ml in a 24-h incubation assay. The lactate dehydrogenase (LDH)-based CytoTox 96TM assay (Promega, Madison, WI) was used to determine cytotoxicity (25). Briefly, 1 × 104 cells/sample, in quadruplicate, were distributed into a 96-well flat-bottom microtiter plate (Costar, Cambridge, MA) and cultured at a low serum concentration (0.1% FBS) 18 h prior to each treatment. After incubation for each different experimental condition, released LDH into the culture supernatants was measured with a 30-min coupled enzymatic assay, which results in the conversion of a tetrazolium salt (INT) into a red formazan product that is read at 490 nm in an automated plate reader (Emax, Molecular Devices, Sunnyvale, CA). Percentage cytotoxicity was calculated using the spontaneous release-corrected OD as follows: % cytotoxicity = (OD of experimental well/OD of maximum release control well) × 100.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-- Total RNA was extracted and purified from ~1 × 106 cells for each experimental condition by a single-step monophasic solution of phenol and guanidine isothiocyanate-chloroform using Trizol® reagent (Life Technologies, Inc.). 1 µg of total RNA was reverse-transcribed to first strand cDNA for 1 h at 42 °C with 200 units of SuperScriptTM II reverse transcriptase and 20 µM random hexamer primers (Life Technologies, Inc.). Amplification of 1/10 of these cDNA by PCR was performed using the following gene-specific primers: TNF-alpha (forward) (5'-AAG CCT GTA GCC CAT GTT GTA GC-3') and TNF-alpha (reverse) (5'-GAA GAC CCC TCC CAG ATA GAT G-3') (342-base pair expected product). Internal control for equal cDNA loading in each reaction was assessed using the following gene-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers: GAPDH sense (5'-GAA CAT CAT CCC TGC CTC TAC TG-3'), GAPDH antisense (5'-GTT GCT GTA GCC AAA TTC GTT G-3') (355-base pair expected product). PCR amplifications of each specific DNA sequence were carried out using the "Hot Start" method using Platinum TaqTM polymerase (Life Technologies, Inc.) followed by a two-step thermal cycling incubations (95 °C/15 s; 60 °C/30 s for 30 cycles and a final extension at 72 °C/10 min). The numbers of PCR cycles were established based on preliminary titration of the relative amount of amplified product for each gene representing the linear phase of the amplification process. The amplified products were resolved on 1.5% agarose gel electrophoresis, and their relative concentrations were assessed by densitometric analysis of the digitized ethidium bromide (EtBr)-stained image, performed on a Macintosh computer (Apple Computer Inc., Cupertino, CA.) using the public domain NIH Image program (developed at the United States National Institutes of Health and available on the Internet).

Nuclear Extract Preparation-- 1 × 106 cultured cells treated under different experimental conditions were washed twice with ice-cold Dulbecco's phosphate-buffered saline (MediaTech, Inc., Herndon, VA). P-40 lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, 0.1 mM EDTA) was added to the top of the washed cells and incubated on ice for 5 min. Lysed cells were collected by gentle pipetting three to four times and transfered to a microcentrifuge tube. Nuclear pellets for each experimental condition were generated by two consecutive centrifugation and washing steps at 1200 rpm. Nuclear pellets were lysed in buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol). Total nuclear protein concentrations were determined using the method of Bradford (26).

Electrophoretic Mobility Shift Assay (EMSA)-- Nuclear protein extracts (2 µg) were assayed for DNA interaction by EMSA as described previously with modifications (27). The double-stranded NF-kappa B consensus binding sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3') oligonucleotide was radiolabeled with [gamma -32P]ATP (ICN Pharmaceuticals, Inc. Costa Mesa, CA.) by incubation with 10 units of T4 polynucleotide kinase (New England Biolabs, Beverly, MA.) and further purified using QIAquick nucleotide removal kit (Qiagen, Valencia, CA.). After the DNA-binding reaction, the samples were resolved on 4-15% Tris-HCl-polyacrylamide minigels (Bio-Rad) and the gels were dried and autoradiographed. Specificity of the DNA-binding reaction was determined by competition assays performed with 100-fold excess of unlabeled NF-kappa B or unrelated oligonucleotide (i.e. AP-1: 5'-GAT CGA ACT GAC CGC CCG CGG CCC GT-3'). The relative concentrations of specific NF-kappa B shifted bands were assessed by densitometric analysis of the digitized autoradiographic images using the NIH Image program described above.

Determination of Intracellular H2O2 Generation-- 1 × 106 cells were cultured in a six-well plate for 18 h in culture medium supplemented with 1% FBS. In some instances, the minimal serum-cultured cells were treated with 500 µM photo-activated SNAP 2 h prior to stimulation with 10 or 100 units/ml TNF-alpha . Intracellular H2O2 levels were evaluated using the fluorescent cell permeable probe, 2',7'-dichlorofluorescein diacetate (H2DCFDA) (Molecular Probes, Inc., Eugene, OR). Then, the culture medium was replaced with Dulbecco's phosphate-buffered saline, pH 7.4, containing 5 µM H2DCFDA. Fluorescence intensity was analyzed on an EPICS® XL-MCL flow cytometer (Beckman Coulter Inc., Fullerton, CA).

Transfections and Reporter Gene System-- The intracellular activation of NF-kappa B was determined by transient transfection of AD10 cells with the pNF-kappa B-d2EGFP reporter vector (CLONTECH, Palo Alto, CA). 7 × 106 cultured cells were transfected with 10 µg of DNA using 60 µl of Lipofectamine reagent (Life Technologies, Inc.) according to the manufacturer's recommendations. Transfected cells were then distributed onto a six-well culture plate and incubated under different experimental conditions. The relative fluorescence intensity was analyzed on an EPICS® XL-MCL flow cytometer.

Statistical Analysis-- The experimental values were expressed as the means ± standard error of the mean (S.E.) for the number of separate experiments indicated in each case. One-way analysis of variance was used to compare variances within groups and among them. Bartlett's tests were used to establish the homogeneity of variance on the basis of the differences among standard deviations (S.D.). Whenever needed, post hoc unpaired multiple comparison tests (Bonferroni's test) and Student's t test were used for comparison between two groups. Significant differences were considered for those probabilities < 5% (p < 0.05).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IFN-gamma -mediated Sensitization of the Human Ovarian Carcinoma AD10 to TNF-alpha -induced Cytotoxicity Is Due, in Part, to the Generation of Nitric Oxide-- To investigate the role of nitric oxide on the sensitization of the human ovarian carcinoma AD10 cell line to TNF-alpha -mediated cytotoxicity, we first stimulated quiescent AD10 cells with IFN-gamma in the presence or absence of 1 mM potent NOS inhibitor L-NMA. The sensitivity of AD10 cells to the cytotoxic effect of increasing concentrations of TNF-alpha (0.01, 0.1, and 1 ng/ml) was evaluated by the release of LDH into the culture medium after 24 h of incubation. Exposure of AD10 cells to IFN-gamma (100 units/ml) for 18 h sensitized the tumor cells to TNF-alpha -mediated cytotoxicity and the degree of sensitization increased with increasing concentrations of TNF-alpha . Sensitization by IFN-gamma was significantly decreased in the presence of 1 mM NOS inhibitor L-NMA (Fig. 1A).


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Fig. 1.   Effect of nitric oxide on the sensitization of AD10 cells to TNF-alpha -induced cytotoxicity. The cytotoxic effect of increasing concentrations of TNF-alpha (0, 0.01, 0.1, and 1 ng/ml) in a 24-h incubation assay was assessed by the LDH release into the culture medium. A, AD10 cells were pretreated with 100 units/ml IFN-gamma for 18 h in the presence or absence of 1 mM NOS inhibitor L-NMA. B, AD10 cells were treated with the NO donor SNAP (0, 10, and 100 µM) 18 h prior to exposure to TNF-alpha . **, p < 0.005; ***, p < 0.001.

To confirm the specific role of nitric oxide in the sensitization of AD10 cells, we assessed the cytotoxic effect of TNF-alpha in the presence of 10 and 100 µM nitric oxide donor SNAP. After incubation for 18 h with different concentrations of SNAP, we observed a significant increase in the sensitivity of AD10 cells to TNF-alpha -mediated cytotoxicity in a 24-h assay that directly correlated with the concentrations of SNAP (Fig. 1B).

Similarly, we have found that IFN-gamma (100 units/ml) sensitized the prostatic adenocarcinoma cell line PC3 to TNF-alpha -mediated cytotoxicity (1 ng/ml) from 5 ± 1.9% to 37 ± 1.2% and the sensitization was blocked by the addition of L-NMA (1 mM) to 20 ± 2.1%. Like IFN-gamma , the use of the NO donor SNAP (100 µM) sensitized PC-3 cells to TNF-alpha (1 ng/ml) cytotoxicity from 1.9 ± 3% to 58 ± 5%.

Nitric Oxide and Pyrrolidine Dithiocarbamate (PDTC) Inhibit TNF-alpha -induced Expression of Endogenous TNF-alpha in AD10 Cells-- The transcription factor NF-kappa B has been demonstrated to tightly regulate the gene expression of TNF-alpha , establishing a self-regulatory loop in tumor cells that secrete TNF-alpha that in turn activates NF-kappa B (28). Furthermore, PDTC has been shown to inhibit TNF-alpha -mediated activation of NF-kappa B in several cell types and in macrophages (29). To demonstrate the specific effect of nitric oxide on the NF-kappa B-mediated expression of TNF-alpha , we incubated AD10 cells with 1, 10, 100, and 500 µM SNAP for 18 h and then stimulated the cells with 100 units/ml TNF-alpha for 4 h. The relative levels of endogenously generated TNF-alpha were assessed by amplification of the specific TNF-alpha cDNA using RT-PCR. The constitutive expression of TNF-alpha by AD10 cells was demonstrated and a significant increased level was observed upon treatment with exogenous TNF-alpha . Moreover, this increased level of TNF-alpha was blocked following treatment of the cells with SNAP (500 µM nitric oxide donor) up to the complete disappearance of the amplified TNF-alpha mRNA (Fig. 2A). These findings suggest that NO inhibits NF-kappa B and consequently down-regulates TNF-alpha mRNA expression. Similar results to those observed with AD10 cells were obtained with the human prostatic adenocarcinoma cell line PC-3. The expression of TNF-alpha messenger RNA in PC-3 was decreased approximately 4-5-fold upon treatment with 500 µM SNAP, suggesting the role of nitric oxide in the NF-kappa B-dependent expression of TNF-alpha .


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Fig. 2.   Role of NO and PDTC on endogenous TNF-alpha gene expression. The relative expression of TNF-alpha mRNA was assessed by RT-PCR. A, AD10 cells were pretreated in the presence or absence of increasing concentrations of the NO donor SNAP (1, 10, 100, and 500 µM) for 18 h and then stimulated with 100 units/ml TNF-alpha for 4 h. B, AD10 cells were pretreated in the presence or absence of increasing concentrations of PDTC (1, 10, 100, and 500 µM) for 18 h and then stimulated with 100 units/ml TNF-alpha for 4 h. Amplification of GAPDH (G-3-PDH) mRNA was used as internal standard control of gene expression for relative comparison.

To confirm the control of NF-kappa B on TNF-alpha expression, we examined the relative levels of expression of endogenous TNF-alpha mRNA after treatment of AD10 cells with 1, 10, 100, and 500 µM PDTC for 18 h followed with TNF-alpha (100 units/ml) stimulation for 4 h. Endogenous TNF-alpha gene expression of TNF-alpha -stimulated cells decreased in the presence of PDTC but was never completely blocked as was observed above following treatment with SNAP (Fig. 2B). These results confirm the role of ROS in the activation of the transcription factor NF-kappa B and the subsequent expression of TNF-alpha .

Nitric Oxide Disrupts the H2O2-dependent Activation of NF-kappa B in AD10 Cells-- To determine whether nitric oxide could interfere with the TNF-alpha -mediated activation of NF-kappa B, we examined the NF-kappa B DNA-binding activity by EMSA. As shown in Fig. 3, nuclear extracts from TNF-alpha -stimulated AD10 cells exhibited an increased binding activity specific for the NF-kappa B heterodimer p65-p50. H2O2 also induced specific NF-kappa B binding activity in AD10 cells after 30 min of incubation. Further, NF-kappa B binding activity was significantly inhibited by the incubation of AD10 cells with 500 µM SNAP for 2 h prior to stimulation with TNF-alpha for 30 min. The impaired NF-kappa B binding activity by SNAP was restored by the addition of H2O2 to similar levels as those detected in the H2O2-stimulated AD10 cells. Thus, these results suggest that the step at which nitric oxide interferes preceded the step at which H2O2 is generated after stimulation of AD10 cells with TNF-alpha .


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Fig. 3.   Effect of NO on the TNF-alpha -mediated NF-kappa B nuclear translocation. Nuclear extracts from AD10 cells pretreated with TNF-alpha in the presence or absence of the NO donor SNAP (500 µM) were analyzed for their NF-kappa B DNA-binding ability using EMSA. Exogenous H2O2 was used as a positive NF-kappa B activator that bypasses the generation of O&cjs1138;2. ***, p < 0.001.

Noteworthy, AD10 cells exhibit a constitutive level of NF-kappa B binding activity that is not affected by nitric oxide (Fig. 3, lanes 1 and 10), whereas in TNF-alpha -stimulated cells the NF-kappa B binding activity decreased below the basal levels in the presence of nitric oxide (Fig. 3, lane 6).

Nitric Oxide Decreases TNF-alpha -dependent Generation of H2O2-- To examine whether nitric oxide affects the generation of H2O2 in AD10 cells stimulated with TNF-alpha , we determined the intracellular generation of H2O2 using the fluorescent cell-permeable probe, H2DCFDA. AD10 cells were incubated in the presence or absence of 500 µM SNAP and then stimulated with 10 and 100 units/ml TNF-alpha , respectively, for 15 min. Fluorescence cytometric analysis of these experimental groups revealed a significant increase in H2O2 levels generated by the TNF-alpha treatment. Incubation of the TNF-alpha -stimulated cells in the presence of SNAP significantly reduced the relative amount of H2O2 generated by these cells (Fig. 4). These data suggest that nitric oxide is affecting the intracellular biogeneration of H2O2 by superoxide dismutase via its chemical interaction with TNF-alpha -induced O&cjs1138;2.


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Fig. 4.   Effect of NO on TNF-alpha -dependent generation of H2O2. Changes in the intracellular H2O2 levels of AD10 cells treated with TNF-alpha in the presence or absence of the NO donor SNAP (500 µM) were assessed by fluorescence flow cytometry. ***, p < 0.001.

Exogenous H2O2 Restored the Nitric Oxide-mediated Blocking of the TNF-alpha -dependent Activation of NF-kappa B-- Nitric oxide has been shown to directly affect the structure of NF-kappa B and decrease its DNA-binding ability due to thiol modification of critical amino acid residues (30). To determine the direct effect of nitric oxide on the activation of NF-kB, we used an enhanced green fluorescent protein-based reporter system driven by four tandem-repeated kappa B responsive elements linked to the thymidine kinase minimal promoter (pNF-kappa B-d2EGFP). We transiently transfected AD10 cells with the pNF-kappa B-d2EGFP reporter vector and then stimulated the cells in the presence or absence of 500 µM SNAP. Cytofluorometric analysis of these cells revealed a significant activation of the reporter gene by TNF-alpha and H2O2, and the extent of activation was a function of the concentrations used. The TNF-alpha -induced activation of the NF-kappa B-dependent reporter gene was significantly decreased in the presence of 500 µM SNAP (Fig. 5), corroborating the findings obtained in the NF-kappa B binding assay. The inhibitory activity of SNAP on the TNF-alpha -induced activation of the NF-kappa B-dependent reporter gene was significantly rescued by stimulation with 200 µM exogenous H2O2 (Fig. 5). These data confirm the inhibitory effect of nitric oxide on the H2O2-dependent activation of NF-kappa B in TNF-alpha -treated AD10 cells. We also noticed that untreated AD10 cells were able to maintain basal levels of NF-kappa B activation that were not inhibited by treatment with nitric oxide, corroborating the findings observed in the binding assay in Fig. 3.


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Fig. 5.   Effect of NO on the TNF-alpha -dependent activation of NF-kappa B. Transiently transfected AD10 cells with the NF-kappa B/2EGFP reporter vector were stimulated with TNF-alpha (10 and 100 units/ml) for 2-3 h in the presence or absence of the NO donor SNAP (500 µM). Exogenous H2O2 was used as a positive NF-kappa B activator that bypasses the generation of O&cjs1138;2. Total mean intensity was determined by fluorescence flow cytometry. Statistical paired comparisons were established between columns 6 and 7 against columns 2 and 3, and columns 8 and 9 against columns 6 and 7. ***, p < 0.001.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The activation of the transcription factor NF-kappa B by TNF-alpha and many other stimuli has been implicated in the development of resistance of tumor cells to a variety of cytotoxic molecules including TNF-alpha (3, 5). NF-kappa B is an oxidative stress-responsive transcription factor that has been shown to respond to small concentrations of exogenous H2O2 or to reactive oxygen species endogenously generated as part of the signaling cascade triggered by many molecules such as TNF-alpha (31-33). We have reported that the IFN-gamma -induced sensitization of the human ovarian carcinoma AD10 cell line to Fas-mediated apoptosis is due in part to the generation of nitric oxide, or its reaction products, by iNOS in these cells (21). In the present study, evidence is presented for the first time that demonstrates that NO also sensitizes tumor cells to TNF-alpha -mediated cytotoxicity. Further, we describe a novel molecular mechanism by which nitric oxide disrupts the H2O2-dependent activation of NF-kappa B resulting in sensitization of the AD10 cells to TNF-alpha cytotoxicity.

The specific role of nitric oxide in tumor biology is not established. A broad spectrum of activities has been assigned to either the physiology or the pathophysiology of nitric oxide in tumor cells (for a review, see Ref. 34). Low output of nitric oxide has been correlated with increased blood flow and new blood vessels feeding the tumor area (35). In addition, the generation of nitric oxide by tumor cells may inhibit the activation and proliferation or may increase the apoptosis of surrounding lymphocytes that can account for the immune suppression accompanying tumor growth (data not shown). Furthermore, high intratumoral output of nitric oxide could inhibit the activation of caspases and therefore antagonizes the pro-apoptotic signals (36, 37). However, the opposite effect has also been observed in many other systems whereby the generation of high output of nitric oxide, either by iNOS induction or by the use of NO donors, inhibits tumor growth and metastasis (38). Therefore, the final outcome of NO-mediated effects would be determined by many factors including the local concentration and sources of nitric oxide and the presence of reactive molecules that might redirect the redox status in the tumor cell.

In the human ovarian carcinoma AD10 cell line stimulated with the pro-inflammatory cytokine IFN-gamma , we observed a markedly increased sensitivity of these tumor cells to the cytotoxic effect of TNF-alpha . IFN-gamma also induces iNOS expression in these cells (21). Sensitization to TNF-alpha was antagonized by the use of the specific NOS inhibitor L-NMA and was mimicked by the use of the NO donor SNAP, confirming the role of nitric oxide in the sensitization process (Fig. 1). Frequently, IFN-gamma treatment alone might not be sufficient to induce iNOS expression in cultured cells. The participation of IFN-gamma in the induction of iNOS is generally directed to the potentiation of the activity of pro-inflammatory cytokines like TNF-alpha , interleukin-1, or bacterial lipopolysaccharide. These cytokines and/or the bacterial lipopolysaccharide have been shown to activate the transcription factor NF-kappa B, setting the basal threshold for the induction of the expression of iNOS that might be enhanced by the action of IFN-gamma (39). We observed that untreated AD10 cells (which constitutively secrete TNF-alpha ) display a constitutive level of activation of NF-kappa B (Fig. 3 and 5). Therefore, the basal activation of NF-kappa B in AD10 cells could explain why the treatment with IFN-gamma alone was sufficient to induce iNOS and subsequently generate nitric oxide.

NF-kappa B has been shown to be a key transcription factor controlling TNF-alpha gene expression in many cells, either as a major activator or synergistically in association with other transcription factors (28). Thus, the significant basal activation of the NF-kappa B in AD10 cells might explain the presence of a constitutive expression of TNF-alpha by these cells (Fig. 2, A and B, last lanes). Moreover, TNF-alpha has been implicated as a survival cytokine used by tumor cells either to control anti-apoptotic mechanisms or promoting cellular proliferation (40-42). Therefore, the maintenance of a self-regulated loop in which the expression of TNF-alpha is perpetuated by the TNF-alpha -mediated basal activation of NF-kappa B could play a major role in the survival and/or proliferation of tumor cells. PDTC has been shown to be a potent and specific inhibitor of the NF-kappa B-mediated expression of TNF-alpha (29, 43). Untreated AD10 cells exhibited a basal expression of TNF-alpha , which was enhanced by stimulation with exogenous TNF-alpha and subsequently inhibited by PDTC (Fig. 2B). Similarly, using the nitric oxide donor SNAP, we were able to completely abrogate the expression of endogenous TNF-alpha (Fig. 2A). In contrast, nitric oxide was unable to block the basal expression of endogenous TNF-alpha in the absence of exogenous stimulation. These results strongly suggest the inhibitory role of nitric oxide on TNF-alpha -induced activation of NF-kappa B and consequently resulting in the disruption of TNF-alpha gene expression.

TNF-alpha induces the generation of ROS that may serve as second messengers in the activation of divergent pathways related to the cell death processes (44-46). Stimulation of many cell types with TNF-alpha results in the generation of intracellular superoxide (O&cjs1138;2) (10). In biological systems, O&cjs1138;2 is immediately reduced by superoxide dismutase to H2O2 or rapidly reacts with NO, generating ONOO- (13). Therefore, decreased amounts of TNF-alpha -generated O&cjs1138;2 will result in a reduced generation of total H2O2. This could subsequently affect the H2O2-dependent activation of NF-kappa B (47). Examining the endogenous generation of H2O2 in TNF-alpha -stimulated AD10 cells, we have found a significant reduction in the total amount of H2O2 being generated in the presence of nitric oxide (Fig. 4). These results strongly suggest the scavenging effect of NO on the O&cjs1138;2 being generated upon TNF-alpha treatment. Alternatively, NO can inhibits O&cjs1138;2 production by the modification of the activity of NADPH oxidase, the main enzyme that generates O&cjs1138;2 within the cell (48, 49).

Further, we have found that the addition of NO donors to TNF-alpha -stimulated AD10 cells inhibited either the DNA binding activity of NF-kappa B (Fig. 3) or its activation (Fig. 5). This inhibition was restored to the normal H2O2-stimulated level by treatment with exogenous H2O2. In contrast, nitric oxide did not affect the NF-kappa B activation in untreated AD10 cells, confirming the previous observation with TNF-alpha gene expression. These results suggest the presence of at least two pathways in the activation of NF-kappa B in AD10 cells that may differ in their sensitivity to H2O2 and the selectivity of nitric oxide to affect just one of these two pathways. The inactivation of NF-kappa B upon NO treatment was not mediated by guanylate cyclase activation since the cGMP analogue 8-bromo-cGMP had no effect on NF-kappa B and we could not block the inhibitory effect of NO on NF-kappa B activation by the use of the guanylate cyclase blocker 1H-(1,2,4)oxadiazolo-[4,3-a]quinoxalin-1-one (data not shown).

Previous reports have implicated the role of nitric oxide on the activation of NF-kappa B. NO has been shown to increase the expression of the NF-kappa B inhibitory subunit Ikappa B or affects its cellular stability by inhibiting protein degradation (50). Due to the rapid generation of H2O2 upon TNF-alpha treatment and the immediate activation NF-kappa B (less than 15 min) in AD10 cells, it is highly unlikely that secondary regulatory factors like the induction of Ikappa B interfered with the rapid NF-kappa B activation. It is likely that, in the long run, a combination of both mechanisms may account for the total inhibitory role of nitric oxide on the TNF-alpha -induced activation of NF-kappa B.

An alternative proposed mechanism implicated in the inhibition of the NF-kappa B activity by NO is via the alteration of critical thiol groups, resulting in the disruption of the NF-kappa B structure and subsequently affecting its DNA-binding ability (30). However, the in vivo situation may be much more complex due to the high concentrations of glutathione and other redox-active proteins within the cell, which may prevent the modification of thiol groups.

In conclusion, our findings suggest that the mechanism by which NO sensitizes the human ovarian carcinoma cell line to TNF-alpha -mediated apoptosis is due to the specific disruption of the TNF-alpha -induced generation of H2O2 and the subsequent inhibition of the NF-kappa B-dependent expression of anti-apoptotic genes. These results can be extended to other solid tumor cells, as observed with the human prostatic adenocarcinoma cell line PC-3. As shown in Fig. 6, the survival autocrine-paracrine loop involving the NF-kappa B-dependent expression of TNF-alpha could be interrupted by the inhibitory activity that nitric oxide exerts on the TNF-alpha -induced activation of NF-kappa B. Furthermore, in an in vivo situation, the exposure of tumor cells to pro-inflammatory cytokines such as IFN-gamma will promote the induction of iNOS by the tumor cells or neighboring lymphocytes and which in turn will result in the generation of nitric oxide. Hence, the endogenously generated or the exogenously provided NO would scavenge the TNF-alpha -generated O&cjs1138;2 and decrease the H2O2-dependent activation of NF-kappa B. Based on these molecular events, a new mechanism of NO-mediated sensitization to apoptosis is revealed.


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Fig. 6.   Schematic representation of possible site of action of NO in the disruption of the TNF-alpha -dependent generation of H2O2 and subsequent inhibition of the activation of NF-kappa B. The autocrine-paracrine loop involving the NF-kappa B-dependent expression of TNF-alpha maintains cell's resistance to apoptotic stimuli and survival. This loop could be interrupted by the inhibitory activity that nitric oxide exerts on the TNF-alpha -induced activation of NF-kappa B. In an in vivo situation, the exposure of tumor cells to pro-inflammatory cytokines, such as IFN-gamma , will promote the induction of iNOS by the tumor cells or neighboring lymphocytes and resulting in the generation of nitric oxide. Hence, the endogenously generated or the exogenously provided NO would scavenge the TNF-alpha -generated O&cjs1138;2 and decrease the H2O2-dependent activation of NF-kappa B. This results in inhibition of NF-kappa B-dependent gene expression of anti-apoptotic factors and sensitization of cells to apoptosis.


    ACKNOWLEDGEMENTS

We thank Dr. Diana Márquez and Dr. Jon Fukuto for their thorough discussion of this study and expert assistance with the technical aspects of the regulation of gene expression and NO-mediated effects, respectively. We are grateful to Chuen-Pei Ng for assistance with the fluorescence cytometry analysis. We also thank Stephanie Louie for secretarial assistance in the preparation of the manuscript.

    FOOTNOTES

* This work was supported in part by the Boiron Research Foundation, the UCLA Gene Therapy Program, the National Council of Science and Technology of Venezuela, and a grant from the Vollmer Foundation (to H. J. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom proofs and reprint requests should be addressed: Dept. of Microbiology, Immunology, and Molecular Genetics, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1747. Tel.: 310-825-2233; Fax: 310-206-3865;E-mail: bbonavida@mednet.ucla.edu.

Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M008471200

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; IFN-gamma , interferon gamma ; iNOS, inducible nitric-oxide synthase; SNAP, S-nitroso-N-acetylpenicillamine; L-NMA, NG-monomethyl-L-arginine; TNF, tumor necrosis factor; LDH, lactate dehydrogenase; RT, reverse transcription; PCR, polymerase chain reaction; PDTC, pyrrolidine dithiocarbamate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; TNFR, tumor necrosis factor receptor; FBS, fetal bovine serum; ROS, reactive oxygen species; H2DCFDA, 2',7'-dichlorofluorescein diacetate..

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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