 |
INTRODUCTION |
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-
B1 as one of the
primary signals in the onset of resistance to many apoptotic stimuli,
particularly TNF-
(3-5).
TNF-
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-
are
mediated by the TNFR1. It has been demonstrated that, upon interaction
with TNF-
, 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-
B (8).
Reactive oxygen species (ROS) have also been implicated in the
signaling pathways initiated by TNF-
. Stimulation of mammalian cells
with TNF-
triggers the generation of various ROS (9, 10). Hence, the
use of antioxidants results in the inhibition of various
TNF-
-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-
(11). These
data support the hypothesis that ROS function as second messengers for
TNF-
-mediated signaling. In biological systems the most important
ROS generated upon TNF-
stimulation are the result of enzymatic
partial reduction of oxygen yielding superoxide (O
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-
signaling via the disruption of
ROS-dependent activation of NF-
B has not been established.
Several lines of evidence showed that resistant tumors could be
sensitized to TNF-
-mediated cytotoxicity by various cytokines or
pharmacological treatments (15-20). Recently, we have reported that
IFN-
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
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-
-mediated signaling by chemically reacting with
O
2. Since can serve as a precursor to
H2O2, which is a proposed activator of the
anti-apoptotic transcription factor NF-
B, the reaction of O
2 with NO will interfere with the activation of NF-
B and
will result in the removal of anti-apoptotic signals and sensitization of the tumor cells to TNF-
cytotoxicity. This study has been designed to test this hypothesis, and the following have been examined:
(a) the molecular mechanism by which IFN-
sensitizes the
human ovarian carcinoma cell line to TNF-
-induced cytotoxicity, (b) the specific role of NO in the disruption of
TNF-
-mediated generation of H2O2, and,
subsequently, (c) the mechanism by which NO can disrupt the
TNF-
-dependent NF-
B activation.
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EXPERIMENTAL PROCEDURES |
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-
(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-
-mediated cytotoxicity was
assessed using recombinant TNF-
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-
(forward) (5'-AAG
CCT GTA GCC CAT GTT GTA GC-3') and TNF-
(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-
B
consensus binding sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3')
oligonucleotide was radiolabeled with [
-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-
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-
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-
. 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-
B was determined by transient transfection of AD10
cells with the pNF-
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 |
IFN-
-mediated Sensitization of the Human Ovarian Carcinoma AD10
to TNF-
-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-
-mediated cytotoxicity, we first stimulated quiescent AD10 cells
with IFN-
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-
(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-
(100 units/ml) for 18 h sensitized the tumor cells to
TNF-
-mediated cytotoxicity and the degree of sensitization increased
with increasing concentrations of TNF-
. Sensitization by IFN-
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- -induced
cytotoxicity. The cytotoxic effect of increasing concentrations of
TNF- (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- 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- . **, p < 0.005; ***,
p < 0.001.
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|
To confirm the specific role of nitric oxide in the sensitization of
AD10 cells, we assessed the cytotoxic effect of TNF-
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-
-mediated cytotoxicity in a 24-h assay that directly correlated with the concentrations of SNAP (Fig. 1B).
Similarly, we have found that IFN-
(100 units/ml) sensitized the
prostatic adenocarcinoma cell line PC3 to TNF-
-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-
, the use of the NO
donor SNAP (100 µM) sensitized PC-3 cells to TNF-
(1 ng/ml) cytotoxicity from 1.9 ± 3% to 58 ± 5%.
Nitric Oxide and Pyrrolidine Dithiocarbamate (PDTC) Inhibit
TNF-
-induced Expression of Endogenous TNF-
in AD10
Cells--
The transcription factor NF-
B has been demonstrated to
tightly regulate the gene expression of TNF-
, establishing a
self-regulatory loop in tumor cells that secrete TNF-
that in turn
activates NF-
B (28). Furthermore, PDTC has been shown to inhibit
TNF-
-mediated activation of NF-
B in several cell types and in
macrophages (29). To demonstrate the specific effect of nitric oxide on the NF-
B-mediated expression of TNF-
, 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-
for 4 h. The
relative levels of endogenously generated TNF-
were assessed by
amplification of the specific TNF-
cDNA using RT-PCR. The
constitutive expression of TNF-
by AD10 cells was demonstrated and a
significant increased level was observed upon treatment with exogenous
TNF-
. Moreover, this increased level of TNF-
was blocked
following treatment of the cells with SNAP (500 µM nitric
oxide donor) up to the complete disappearance of the amplified TNF-
mRNA (Fig. 2A). These
findings suggest that NO inhibits NF-
B and consequently
down-regulates TNF-
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-
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-
B-dependent expression of TNF-
.

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Fig. 2.
Role of NO and PDTC on endogenous
TNF- gene expression. The relative
expression of TNF- 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- 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- for 4 h. Amplification of GAPDH
(G-3-PDH) mRNA was used as internal standard control of
gene expression for relative comparison.
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|
To confirm the control of NF-
B on TNF-
expression, we examined
the relative levels of expression of endogenous TNF-
mRNA after
treatment of AD10 cells with 1, 10, 100, and 500 µM PDTC for 18 h followed with TNF-
(100 units/ml) stimulation for
4 h. Endogenous TNF-
gene expression of TNF-
-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-
B and the subsequent expression of
TNF-
.
Nitric Oxide Disrupts the
H2O2-dependent Activation of
NF-
B in AD10 Cells--
To determine whether nitric oxide could
interfere with the TNF-
-mediated activation of NF-
B, we examined
the NF-
B DNA-binding activity by EMSA. As shown in Fig.
3, nuclear extracts from
TNF-
-stimulated AD10 cells exhibited an increased binding activity
specific for the NF-
B heterodimer p65-p50.
H2O2 also induced specific NF-
B binding
activity in AD10 cells after 30 min of incubation. Further, NF-
B
binding activity was significantly inhibited by the incubation of AD10
cells with 500 µM SNAP for 2 h prior to stimulation
with TNF-
for 30 min. The impaired NF-
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-
.

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Fig. 3.
Effect of NO on the
TNF- -mediated NF- B
nuclear translocation. Nuclear extracts from AD10 cells pretreated
with TNF- in the presence or absence of the NO donor SNAP (500 µM) were analyzed for their NF- B DNA-binding ability
using EMSA. Exogenous H2O2 was used as a
positive NF- B activator that bypasses the generation of O 2.
***, p < 0.001.
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|
Noteworthy, AD10 cells exhibit a constitutive level of NF-
B binding
activity that is not affected by nitric oxide (Fig. 3, lanes
1 and 10), whereas in TNF-
-stimulated cells
the NF-
B binding activity decreased below the basal levels in the
presence of nitric oxide (Fig. 3, lane 6).
Nitric Oxide Decreases TNF-
-dependent Generation of
H2O2--
To examine whether nitric oxide
affects the generation of H2O2 in AD10 cells
stimulated with TNF-
, 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-
, respectively, for 15 min. Fluorescence cytometric
analysis of these experimental groups revealed a significant increase
in H2O2 levels generated by the TNF-
treatment. Incubation of the TNF-
-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-
-induced O
2.

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Fig. 4.
Effect of NO on
TNF- -dependent generation of
H2O2. Changes in the intracellular
H2O2 levels of AD10 cells treated with TNF-
in the presence or absence of the NO donor SNAP (500 µM)
were assessed by fluorescence flow cytometry. ***, p < 0.001.
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Exogenous H2O2 Restored the Nitric
Oxide-mediated Blocking of the TNF-
-dependent Activation
of NF-
B--
Nitric oxide has been shown to directly affect the
structure of NF-
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
B responsive elements linked to the thymidine kinase
minimal promoter (pNF-
B-d2EGFP). We transiently transfected AD10
cells with the pNF-
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-
and
H2O2, and the extent of activation was a
function of the concentrations used. The TNF-
-induced activation of
the NF-
B-dependent reporter gene was significantly
decreased in the presence of 500 µM SNAP (Fig.
5), corroborating the findings obtained
in the NF-
B binding assay. The inhibitory activity of SNAP on the
TNF-
-induced activation of the NF-
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-
B in TNF-
-treated AD10 cells. We also noticed that untreated
AD10 cells were able to maintain basal levels of NF-
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- -dependent activation of
NF- B. Transiently transfected AD10 cells
with the NF- B/2EGFP reporter vector were stimulated with TNF- (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- B activator that bypasses the generation of
O 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 |
The activation of the transcription factor NF-
B by TNF-
and
many other stimuli has been implicated in the development of resistance
of tumor cells to a variety of cytotoxic molecules including TNF-
(3, 5). NF-
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-
(31-33). We have reported that the IFN-
-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-
-mediated
cytotoxicity. Further, we describe a novel molecular mechanism by which
nitric oxide disrupts the
H2O2-dependent activation of
NF-
B resulting in sensitization of the AD10 cells to TNF-
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-
, we observed a markedly increased
sensitivity of these tumor cells to the cytotoxic effect of TNF-
.
IFN-
also induces iNOS expression in these cells (21). Sensitization
to TNF-
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-
treatment alone might not be sufficient to
induce iNOS expression in cultured cells. The participation of IFN-
in the induction of iNOS is generally directed to the potentiation of
the activity of pro-inflammatory cytokines like TNF-
, interleukin-1,
or bacterial lipopolysaccharide. These cytokines and/or the bacterial
lipopolysaccharide have been shown to activate the transcription factor
NF-
B, setting the basal threshold for the induction of the
expression of iNOS that might be enhanced by the action of IFN-
(39). We observed that untreated AD10 cells (which constitutively
secrete TNF-
) display a constitutive level of activation of NF-
B
(Fig. 3 and 5). Therefore, the basal activation of NF-
B in AD10
cells could explain why the treatment with IFN-
alone was sufficient
to induce iNOS and subsequently generate nitric oxide.
NF-
B has been shown to be a key transcription factor controlling
TNF-
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-
B in AD10
cells might explain the presence of a constitutive expression of
TNF-
by these cells (Fig. 2, A and B,
last lanes). Moreover, TNF-
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-
is perpetuated by the TNF-
-mediated basal
activation of NF-
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-
B-mediated expression of TNF-
(29,
43). Untreated AD10 cells exhibited a basal expression of TNF-
,
which was enhanced by stimulation with exogenous TNF-
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-
(Fig. 2A). In contrast,
nitric oxide was unable to block the basal expression of endogenous
TNF-
in the absence of exogenous stimulation. These results strongly
suggest the inhibitory role of nitric oxide on TNF-
-induced
activation of NF-
B and consequently resulting in the disruption of
TNF-
gene expression.
TNF-
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-
results in the generation of intracellular superoxide (O
2)
(10). In biological systems, O
2 is immediately reduced by
superoxide dismutase to H2O2 or rapidly reacts
with NO, generating ONOO
(13). Therefore, decreased
amounts of TNF-
-generated O
2 will result in a reduced
generation of total H2O2. This could
subsequently affect the
H2O2-dependent activation of
NF-
B (47). Examining the endogenous generation of
H2O2 in TNF-
-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
2 being generated upon TNF-
treatment.
Alternatively, NO can inhibits O
2 production by the
modification of the activity of NADPH oxidase, the main enzyme that
generates O
2 within the cell (48, 49).
Further, we have found that the addition of NO donors to
TNF-
-stimulated AD10 cells inhibited either the DNA binding activity of NF-
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-
B activation in untreated AD10
cells, confirming the previous observation with TNF-
gene
expression. These results suggest the presence of at least two pathways
in the activation of NF-
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-
B upon NO treatment was not mediated by guanylate cyclase
activation since the cGMP analogue 8-bromo-cGMP had no effect on
NF-
B and we could not block the inhibitory effect of NO on NF-
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-
B. NO has been shown to increase the expression of
the NF-
B inhibitory subunit I
B or affects its cellular stability by inhibiting protein degradation (50). Due to the rapid generation of
H2O2 upon TNF-
treatment and the immediate
activation NF-
B (less than 15 min) in AD10 cells, it is highly
unlikely that secondary regulatory factors like the induction of I
B
interfered with the rapid NF-
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-
-induced activation of
NF-
B.
An alternative proposed mechanism implicated in the inhibition of the
NF-
B activity by NO is via the alteration of critical thiol groups,
resulting in the disruption of the NF-
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-
-mediated apoptosis is due to the specific disruption of the TNF-
-induced generation of H2O2 and the subsequent
inhibition of the NF-
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-
B-dependent expression of TNF-
could be
interrupted by the inhibitory activity that nitric oxide exerts on the
TNF-
-induced activation of NF-
B. Furthermore, in an in
vivo situation, the exposure of tumor cells to pro-inflammatory
cytokines such as IFN-
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-
-generated
O
2 and decrease the
H2O2-dependent activation of
NF-
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- -dependent generation of
H2O2 and subsequent inhibition of the
activation of NF- B. The
autocrine-paracrine loop involving the NF- B-dependent
expression of TNF- 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- -induced activation of NF- B.
In an in vivo situation, the exposure of tumor cells to
pro-inflammatory cytokines, such as IFN- , 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- -generated
O 2 and decrease the
H2O2-dependent activation of
NF- B. This results in inhibition of NF- B-dependent
gene expression of anti-apoptotic factors and sensitization of cells to
apoptosis.
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