1 Departments of Internal Medicine and the Cannon Research Center, Carolinas Medical Center, Charlotte 28232; 2 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham 27710; 4 National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and 3 Division of Respiratory, Critical Care, and Occupational (Pulmonary) Medicine, University of Utah, Salt Lake City, Utah 84132
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ABSTRACT |
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The transcription factor nuclear factor-B (NF-
B) is
constitutively activated in malignancies from enhanced activity of inhibitor of NF-
B (I
B) kinase, with accelerated I
B
degradation. We studied whether redox signaling might stimulate these
events. Cultured melanoma cells generated superoxide anions
(O
) without serum stimulation. O
generation was reduced by the NAD(P)H:quinone oxidoreductase (NQO)
inhibitor dicumarol and the quinone analog capsaicin, suggesting that
electron transfer from NQO through a quinone-mediated pathway may be an important source of endogenous reactive oxygen species (ROS) in tumor
cells. Treatment of malignant melanoma cells with the
H2O2 scavenger catalase, the sulfhydryl donor
N-acetylcysteine, the glutathione peroxidase mimetic
ebselen, or dicumarol decreased NF-
B activation. Catalase,
N-acetylcysteine, ebselen, dicumarol, and capsaicin also
inhibited growth of melanoma and other malignant cell lines. These
results raise the possibility that ROS produced endogenously by
mechanisms involving NQO can constitutively activate NF-
B in an
autocrine fashion and suggest the potential for new antioxidant
strategies for interruption of oxidant signaling of melanoma cell growth.
nuclear factor-B; superoxide anion; hydrogen peroxide; tumor; neoplasm; dicumarol
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INTRODUCTION |
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THE INDUCIBLE
PLEIOTROPIC transcription factor nuclear factor-B (NF-
B) is
well recognized to play a pivotal role in the control of cellular
inflammation. Now, a growing body of evidence points to an important
role for NF-
B in the growth of malignancies. Constitutive nuclear
NF-
B activation has been recently found important for proliferation
of malignant melanoma (53), Hodgkin's disease
(5), and carcinomas of the breast
(56), ovary (12), colon (12),
lung (7), head and neck (21), and pancreas (64). Repression of NF-
B functionally interferes with
normal and transformed cell proliferation (29, 32), and
inhibition of NF-
B by antisense strategies (28) or by
overexpression of the NF-
B inhibitor I
B
(5, 7, 21,
56) blocks tumor cell growth. Also, activation of NF-
B has
been linked with resistance of tumors to tumor necrosis factor-
(TNF-
)-induced apoptosis, anti-cancer chemotherapy, and
radiation (9, 62, 63). Thus NF-
B has emerged as a
critically important regulator of transcription in neoplasms.
In unstimulated normal cells, NF-B resides in the cytoplasm
as a dimeric protein complex bound to an inhibitor protein, designated I
B
(54). Agonist stimulation activates I
B kinase,
which phosphorylates serines-32 and -36 near the amino terminus of
I
B
(3, 57), targeting the inhibitor for
ubiquitination and proteolytic degradation by the 26S
proteasome (46). The removal of I
B
unmasks
the nuclear localization signal (10), allowing the NF-
B
complex to translocate to the nucleus, where it binds to its respective nucleotide sequence and transcriptionally regulates expression or
repression of target genes. In most normal cell types, except for
certain neurons (33) and mature B lymphocytes
(39), nuclear NF-
B activity is observed only
transiently in response to inducers, including phorbol esters, viral
transactivators, and cytokines, which might all act through a common
mechanism involving the synthesis of reactive oxygen species as
proximate messengers (52). In cultured melanoma cell
lines, constitutive activation of NF-
B has been causally related to
enhanced degradation of I
B
(53) from elevated
activity of I
B kinase (19). Thus the constitutive activation of NF-
B found in a number of tumors (5, 7, 12, 21,
53, 56, 64) may involve intermediate signaling events that are
parallel to those occurring during activation of NF-
B in normal cell
lines (3, 57). Human tumor cells produce substantial amounts of reactive oxygen species spontaneously (18, 41, 60). Mitogenic signaling through both Ras (30) and
Rac (31) is mediated by superoxide anion
(O
) production, and transfection with the
superoxide-generating oxidase mox-1 transforms normal cells
(58). Therefore, we determined whether reactive oxygen
species might also mediate constitutive NF-
B activation in malignant
melanoma cell lines. Our findings suggest that endogenous redox stress
is likely responsible for signaling NF-
B activation in malignant
melanoma cells and that scavengers of reactive oxygen species,
especially of H2O2, are potent inhibitors of
tumor cell proliferation. Moreover, the enzymatic source of redox
stress promoting constitutive NF-
B activation appears to be
NAD(P)H:quinone oxidoreductase (NQO), which is overexpressed in many
tumors (11) and can reduce membrane ubiquinone
(35) for redox cycling with molecular oxygen to generate
O
at the plasma membrane, where stimulation of
NF-
B activation occurs.
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MATERIALS AND METHODS |
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Materials.
Human malignant cell lines were obtained from American Type Culture
Collection (Rockville, MD). RPMI medium 1640, Dulbecco's modified
Eagle's medium, Leibovitz's L-15 medium, HEPES,
antibiotic-antimycotic (10,000 units penicillin, 10,000 µg
streptomycin, and 25 µg amphotericin B/ml), and trypsin-EDTA solution
were purchased from the GIBCO BRL division of Life Technologies (Grand
Island, NY). Fetal bovine serum (FBS) was purchased from HyClone
Laboratories (Logan, UT). Rabbit polyclonal supershift antibodies for
electrophoretic mobility shift assays (EMSAs) and for cyclin D1 were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and
phosphospecific antibodies for immunoassay detection of the
phosphorylated form of IB
were purchased from New England Biolabs
(Beverly, MA). Peroxidase-labeled donkey polyclonal anti-rabbit IgG was
from Amersham Life Sciences (Buckinghamshire, England, UK). EMSA
supplies, including DNA probes, were purchased from Promega (Madison,
WI). Protease inhibitors were from Boehringer Mannheim (Indianapolis,
IN). All other materials were purchased from Sigma Chemical (St. Louis,
MO) unless specified.
Culture of malignant cell lines. Melanoma cell lines CRL 1585 and CRL 1619 were cultured in RPMI 1640 with 10% FBS and passed with nonenzymatic Cell Dissociation Solution (Sigma). LNCaP.FGC prostate adenocarcinoma cells were also cultured in RPMI 1640 with 10% FBS but passed with 0.05% trypsin and 0.53 mM EDTA. The adenosquamous lung carcinoma NCI-H596 cell line was grown in RPMI 1640 supplemented with 10% FBS, 10 mM HEPES, and 1.0 mM sodium pyruvate and passed with trypsin/EDTA. All of the above were grown in a 37°C humidified environment containing 5% CO2-95% air. The breast carcinoma cell line MDA-MB-453 was grown in a 37°C humidified environment with free gas exchange with atmospheric air using Leibovitz's L-15 medium with 2 mM L-glutamine and 10% FBS and was passed with trypsin/EDTA.
Cell culture treatments.
The effect of antioxidants on nuclear activation of NF-B was studied
by incubating near-confluent (70%) cell cultures with antioxidant
treatments for 1-48 h. Nuclear protein was harvested, and EMSAs
were performed using DNA consensus-binding sequences. Nuclear
translocation of the p65 component of NF-
B was studied by
immunoperoxidase staining, as outlined in Immunohistochemical localization of NF-
B. The effect of antioxidants on
expression of phosphorylated I
B
was studied by incubating
near-confluent cell cultures with antioxidant treatments for 15 min to
48 h. Cells were lysed and protein expression levels were measured
by immunoblot assay using a phosphospecific antibody for phosphorylated I
B
.
EMSAs.
Nuclear protein was isolated and DNA binding reactions were performed
as previously described in detail (34). Monolayers were
washed twice in cold Dulbecco's phosphate buffered saline (DPBS) and
equilibrated 10 min on ice with 0.7 ml cold cytoplasmic extraction
buffer (CEB), consisting of (in mM) 10 Tris, pH 7.9, 60 KCl, 1 EDTA, 1 dithiothreitol (DTT), with protease inhibitors (PI; 1 mM Pefabloc, 50 µg/ml antipain, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 40 µg/ml
bestatin, 3 µg/ml E-64, and 100 µg/ml chymostatin). The detergent
Nonidet P-40 (NP-40) was added to a final concentration of 0.1%, and
cells were dislodged with a cell scraper. Nuclei were pelleted by
centrifugation and washed with CEB/PI. Nuclei were then incubated for
20 min on ice in nuclear extraction buffer (NEB; 20 mM Tris, pH 8.0, 400 mM NaCl, 1.5 mM MgCl2, 1.5 mM EDTA, 1 mM DTT, and 25%
glycerol) with PI, spun briefly to clear debris, and stored at 80°C
until performance of EMSAs. EMSAs were performed using the consensus
binding oligonucleotides 5'-AGTTGAGGGGACTTTCCCAG-GC-3' and
3'-TCAACTCCCCTGAAAGGGTCCG-5' for the p50 component of NF-
B (Promega,
Madison, WI), end-labeled by phosphorylation with
[
-32P]ATP and T4 polynucleotide kinase. DNA-protein
binding reactions were performed with 2 µg of nuclear protein (as
determined by the Pierce method) and 50-100,000 cpm of
32P end-labeled double-stranded DNA probe in 10 mM
Tris · HCl, pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 1 mM
MgCl2, 50 µg/ml poly(dI-dC), and 4% glycerol. All
components of the binding reaction, with the exception of labeled
probe, were combined and incubated at room temperature for 10 min
before addition of labeled probe and incubated for an additional 20 min. Competition experiments were performed with 10× unlabeled
wild-type oligonucleotide sequences for NF-
B added before labeled
probe. Supershift assays were performed by adding 1.0 µg of
supershift-specific antibodies for p65, p50, p52, Rel B, or c-Rel
components of NF-
B and incubated at room temperature for 30 min or
overnight at 4°C before adding the probe. Samples were
electrophoresed on a 5% nondenaturing polyacrylamide gel in
Tris-glycine-EDTA (120 mM glycine and 1 mM EDTA in 25 mM Tris, pH 8.5)
buffer. Gels were dried and analyzed by autoradiography at
80°C
using an image intensifier screen. Densitometry of bands was performed
using Kodak Digital Science 1D image analysis software (Eastman Kodak,
Rochester, NY).
Immunohistochemical localization of NF-B.
Cells grown on sterile coverslips and treated with catalase (3,000 U/ml), apocynin (150 µg/ml), dicumarol (250 µM), or DPBS or DMSO
vehicles for 24 h were fixed for 20 min on ice with 4% paraformaldehyde in DPBS with protease inhibitors, PI [10 µl/ml of
Sigma protease inhibitor cocktail, containing
4-(2-aminoethyl)benzensulfonyl fluoride, pepstatin A,
trans-epoxysuccinyl-L-leucylamindo-(guanidino)butane (E-64), bestatin, leupeptin, and aprotinin]. Cells were permeabilized by treating for 2 min with 0.1% NP-40 in DPBS/PI, washed once with
cold DPBS, and fixed as before for 10 min. Coverslips were incubated in
3% hydrogen peroxide for 30 min to suppress any remaining peroxidase
and were washed three times in cold DPBS. The permeabilized and fixed
cells were blocked for 2 h with 2% BSA in DPBS on ice and
incubated overnight at 4°C with 1 µg/ml of anti-p65 antibody (Santa
Cruz) diluted in 0.1% BSA/DPBS. Unbound anti-p65 was washed away with
2% BSA/DPBS, and bound antibody was stained by incubation with
biotinylated goat anti-rabbit immunoglobulin diluted 1:50 in 0.1%
BSA/DPBS for 45 min on ice. Excess secondary antibody was washed away
by three washes with 2% BSA/DPBS on ice. After the cells were washed,
they were incubated with a streptavidin-biotin-peroxidase complex at
room temperature for 1 h, washed again, and incubated in 0.03%
wt/vol 3-3'-diaminobenzidine with 0.003% vol/vol hydrogen peroxide until a brown reaction product could be seen. Cells were then
counterstained with eosin and mounted on glass slides before viewing
under light microscopy.
Immunoassay for phosphorylated IB
and cyclin D1.
Cells were lysed, and proteins were isolated and quantitated by
immunoassay as previously detailed (13). Cells were placed on ice, washed twice with cold DPBS, scraped into 0.5 ml boiling buffer
[10% (vol/vol) glycerol and 2% (wt/vol) SDS in 83 mM Tris, pH 6.8]
to which 50 mM DTT had been added as a reducing agent, and sheared by
four passages through a pipette. Aliquots were removed for protein
determination, using the bicinchoninic acid (BCA) protein assay
(Pierce). After 10%
-mercaptoethanol and 0.05% bromphenol blue
were added, lysates were boiled for 5 min and stored at
80°C until
immunoblotting was performed. Proteins in defrosted samples were
separated by SDS-PAGE on 12% polyacrylamide gels (15 µg
protein/lane) and electrotransferred to 0.45 µm Hybond ECL
nitrocellulose membranes (Amersham Life Sciences) using the wet
transblot method in transfer buffer [0.025 M Tris, 0.192 M glycine,
2.6 mM SDS, and 20% (vol/vol) methanol; pH 8.8] at 100 V for 1 h. In samples assayed for phosphorylated I
B
, blots were blocked
for 2 h at room temperature with blocking buffer [PBS with 0.1%
Tween 20 and 5% fat-free milk powder (Carnation, Glendale, CA)].
After the blots were rinsed five times for 5 min each in PBS containing
0.1% Tween 20, they were incubated overnight at 4°C with primary
rabbit polyclonal phosphospecific antibodies for the phosphorylated
form of the NF-
B inhibitor I
B
, diluted 1:1,000 in PBS with
0.1% Tween 20 and 5% BSA. After the blots were rinsed again as above,
they were incubated for 1 h at room temperature with horseradish
peroxidase (HRP)-conjugated secondary antibody diluted 1:5,000 in
blocking buffer. Immunoblots were rinsed again as above and detected
using an enhanced chemiluminescence method (ECL Western blotting
detection system; Amersham Life Science) and autoradiography.
Densitometry was performed as above.
Measurement of GRO- and IL-8 expression.
GRO-
and IL-8 were measured in culture medium from untreated and
ebselen- or dicumarol-treated cells using commercial ELISA assays
purchased from R&D Systems (Minneapolis, MN). This assay could not be
used to detect GRO-
production by catalase-treated cells because of
interference in the peroxidase-based ELISA by catalase in the cell supernatant.
Measurement of proliferation in cell cultures. Proliferation of cultured cells was quantitated using a previously reported colorimetric method based on metabolic reduction of the soluble yellow tetrazolium dye MTT to its insoluble purple formazan by the action of mitochondrial succinyl dehydrogenase (13). This assay empirically distinguishes between dead and living cells. For proliferation studies, cells were seeded into 24-well uncoated plastic plates (Costar) at 15,000-50,000 cells per well and cultured with respective media and mitogens. After 24-96 h, medium was removed, cells were washed twice with 1 ml of sterile Dulbecco's modified PBS without Ca2+ or Mg2+ (DPBS), the medium was replaced with 1 ml/well fresh medium containing 100 µg/ml MTT, and plates were incubated an additional hour. MTT-containing medium was removed, 0.5 ml DMSO was added to each well, and the absorbance of the solubilized purple formazan dye was measured at 540 nm. A total of four to six wells was studied at each treatment condition. Preliminary studies were performed with 50-200 µg/ml MTT incubated for 15 min to 3 h to determine the optimum concentration and incubation time at which the rate of conversion was linear and proportional to the number of cells present. The absorbance of the MTT formazan reduction product (A540) correlated with cell numbers counted by hemocytometer with R2 = 0.99. In some experiments, the MTT assay and responses to mitogens and inhibitors were also confirmed by performing cell counts on 10 random fields/well of Giemsa-modified Wright's stained monolayers viewed at 40 power using a 0.01-cm2 ocular grid.
Measurement of cytotoxicity and apoptosis. To assess for cytotoxicity, near-confluent cells cultured in 24-well plates were exposed to antioxidants or withdrawn from serum for 24 h. Medium was removed and replaced with DPBS containing 0.1% trypan blue. Cell death was assessed by counting the average number of trypan blue positive cells in five random fields counted of eight separate wells at 40 power using a 0.01-cm2 ocular grid. To assess for apoptosis, cells were grown on glass slides in the presence of 10% FBS and treated with 3,000 U/ml catalase for 24 h. Apoptosis was studied by two methods. DNA cleavage was assessed by terminal deoxynucleotidyl transferase (TdT)-dependent 3'-OH fluorescein end-labeling of DNA fragments, using a Fluorescein-FragEL DNA fragmentation detection kit (Oncogene Research Products, Cambridge, MA). DNase-treated fixed cells were used as a positive control. Apoptosis was also evaluated by fluorescent-labeled annexin V staining of phosphatidylserine translocated to membrane surface. Externalized phosphatidylserine was stained using the Annexin-V-FLUOS staining kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's instructions. Positive annexin V labeling was induced by treatment of cells for 24 h with 1 µM staurosporine.
Electron Microscopy. Cells were detached with trypsin/EDTA, fixed in 2.5% glutaraldehyde, postfixed in 1.0% osmium tetroxide, dehydrated sequentially in ethanol and propylene oxide, and embeded in spurr resin. Thin sections were stained sequentially with uranyl acetate and Sato's triple lead stain, and viewed using a Phillips CM10 transmission electron microscope.
DNA cell cycle measurements. To study the effect of antioxidant treatments on the DNA cell cycle, cells were grown to near confluence in 25-cm2 plastic flasks and treated for 24 h. Cells were typsinized, washed twice in cold DPBS with 1 mM EDTA and 1% BSA, fixed 30 min in ice-cold 70% ethanol, and stained by incubation for 30 min at 37°C in a 10 µg/ml solution of propidium iodide in DPBS and 1 mg/ml RNase A. DNA cell cycle measurements were made using a FACStarPLUS Flow Cytometer (Becton-Dickinson, San Jose, CA).
Measurement of reactive oxygen species.
O generation was measured by the technique of
superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c (24), employing a modification allowing
absorbance reading with an automatic enzyme immunoassay reader
(47). Confluent cells grown on 24-well plates were washed
with DPBS and incubated in 5% CO2-95% air at 37°C with
160 µM ferricytochrome c in a total volume of 550 µl of
sodium bicarbonate-containing Krebs-Heinseleit buffer (44)
or Hanks' balanced salt solution (HBSS), with and without copper-zinc
SOD (1,000 U/ml). The absorbance of each well was measured at 550 nm
initially and 3-24 h later using an ELx800 ultraviolet (UV)
automated microplate reader (Biotek Instruments, Highland Park, VT).
Monolayers were then washed with DPBS, and cell protein was measured
using the BCA protein assay (Pierce). O
generation,
normalized to cell protein, was computed from the Beer-Lambert
relationship (6) as the quotient of SOD-inhibitable
increase absorbance over time divided by the difference between
the molar extinction coefficients for ferricytochrome c
and ferrocytochrome c (2.1 × 104
M
1 · cm
1) (24).
In some experiments, the following inhibitors of major oxidases were
added to dissect potential sources of O
generation:
the quinone analog capsaicin
(8-methyl-N-vanillyl-6-noneamide, 100 µM), the NQO
inhibitor dicumarol (250 µM), the xanthine oxidase inhibitor
allopurinol (1 mM), the cyclooxygenase inhibitor indomethacin (10 µg/ml), the cytochrome P-450 inhibitor cimetidine (300 µM), the nitric oxide synthase inhibitor
N
-nitro-L-arginine methyl ester
(LNAME; 100 µM), and the mitochondrial respiratory chain
inhibitor rotenone (2 µM).
Determination of oxidase activities and levels of oxidase components. Xanthine dehydrogenase/oxidase (XDH/XO) activity was measured using the spectrofluorometric assay described by Beckman et al. (8). Briefly, monolayers were washed twice in ice-cold DPBS, scraped, and frozen in liquid nitrogen. The cell pellet was sonicated in 1 ml of buffer containing 0.1 mM EDTA, 10 mM DTT, and 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in 50 mM phosphate buffer, pH 7.4. The cell lysates were centrifuged at 18,000 g for 30 min at 4°C. The supernatant was diluted to 2 ml with 50 mM phosphate buffer containing 0.1 mM EDTA, pH 7.4. Fluorescence was monitored at 390 nm with the excitation wavelength set at 345 nm. After achieving a stable baseline, 20 µl of 1 mM pterin were added, and the reaction was observed for 20 min to assay XO activity. Subsequently, 20 µl of 1 mM methylene blue were added as an electron accceptor to assay total XDH/XO activity, and the reaction was observed for 20 min.
To probe for presence of p22 and gp91phox components to the putative analog of neutrophil NAD(P)H oxidase and the newly described mox-1 (58), also known as NOH-1L (4), total RNA was isolated from cells by the method of Chomzynski and Sacchi (15). The RNA concentration was determined spectrophotometrically, and 5 µg were used for reverse transcription employing a standard protocol with Moloney murine leukemia virus reverse transcriptase. Excess RNA was digested with 2 µg DNAse-free RNAse (Boehringer Mannheim) and incubated at 37°C for 5 min. The reaction was extracted with phenol-chloroform and precipitated with ethanol atStatistical analysis. Data are expressed as means ± SE for a minimum number of four observations, unless otherwise indicated. Differences between two groups were compared using the Student's t-test. Differences between multiple groups were compared using one-way analysis of variance. The post hoc test used was the Newman-Keuls multiple-comparison test. Two-tailed tests of significance were employed. Significance was assumed at P < 0.05.
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RESULTS |
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Antioxidants reduce constitutive activation of NF-B in malignant
human cell lines.
Constitutive activation of NF-
B has been previously reported in
malignant melanoma cell lines (19, 53). M1619 melanoma cells also consistently exhibited constitutive DNA binding activity for
NF-
B in nuclear protein (Fig.
1, A and B, lane
1). Several distinct bands were observed, all of which were
eliminated by addition of excess specific unlabeled NF-
B consensus
oligonucleotides to the binding reaction (Fig. 1B, lane
2). Supershift experiments demonstrated that the second
band (Fig. 1A, arrow) contained p65 (Fig. 1A, lane
2) and p50 (Fig. 1A, lane 3) NF-
B components, but
not p52, Rel-B, or c-Rel (Fig. 1A, lanes 4-6). Identity
of proteins binding in the other bands is presently unclear.
Constitutive nuclear translocation of NF-
B was confirmed
immunohistochemically by intense staining for p65 in M1619 nuclei (Fig.
2A). Treatment of cells for
24 h with the antioxidants catalase or N-acetylcysteine (NAC) or the glutathione peroxidase mimetic (55) ebselen
substantially reduced constitutive NF-
B DNA binding activity in
nuclear extracts (Fig. 1, C and D). Furthermore,
exposure of cells to catalase for 24 h also essentially eliminated
immunohistochemical staining for p65 in cell nuclei (Fig.
2B). In addition, catalase treatment for 24 h also
suppressed constitutive nuclear DNA binding activity for NF-
B in
M1585 melanoma cells. These results suggest that constitutive nuclear
activation of NF-
B in malignant melanoma cell lines may be the
consequence of endogenous redox stress.
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Antioxidants are antiproliferative against malignant human cell
lines.
Inhibiting NF-B activation in tumor cells has been shown to reduce
cellular proliferation. We therefore studied whether the same
antioxidants that decreased constitutive activation of NF-
B also
inhibited growth of malignant cell lines. At concentrations we have
previously reported to inhibit growth of cultured human airway smooth
muscle (13), NAC and catalase reduced proliferation of
M1619 melanoma cells when added to culture medium (Fig.
5A). In contrast, copper-zinc
SOD had no effect on cell growth. Growth inhibition from catalase was
dose dependent (Fig. 5A), was shared by a variety of
catalase preparations from different sources (data not shown), and was
eliminated by protein inactivation (Fig. 5B). Catalase
treatment did not appear to produce apoptosis. Catalase did not
result in externalization of phosphatidylserine to the plasma membrane
surface, studied by fluorescent-labeled annexin V staining (Fig.
6,
A-C) and did not cause DNA fragmentation, studied by
TdT-dependent 3'-OH fluorescein end-labeling of DNA fragments (data not
shown). Also, catalase-treated M1619 cells demonstrated no electron
microscopic evidence of membrane blebbing or condensation of chromatin
(Fig. 6, E vs. D), but catalase did cause
prominent vacuolization of the cell cytoplasm (Fig. 6, E vs.
D) and significantly decreased trypan blue dye exclusion
(1.4 ± 0.2 for vehicle-treated control cells vs. 10.1 ± 0.8 trypan blue positive cells/field for confluent cells treated 24 h
with 3,000 U/ml catalase; P < 0.01). M1619
proliferation was also dramatically reduced by ebselen (Fig.
5C). Catalase and ebselen were antiproliferative even if
added 24 h after melanoma cells were plated (Fig. 5D) and were effective against a wide range of cultured malignant cells,
including carcinomas of the lung, prostate, and breast (Table
1). Together, these results indicate that
reactive oxygen species may be important signaling molecules for growth
of malignant cell lines and suggest that the proximate growth-signaling
form of reactive oxygen may be H2O2.
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Antioxidants reduce polyploidy and increase S-phase fraction in
M1619 cells.
Antioxidant treatments that reduced constitutive activation of NF-B
also had profound effects on the cell cycle. Results of DNA cell cycle
analysis of M1619 cells are shown in Fig.
7. Untreated M1619 melanoma cells are a
rapidly proliferating, desynchronized malignant line composed of both
diploid and tetraploid cells. Over 30% of cells are tetraploid (Fig.
7A). Treatment with catalase for 24 h (Fig.
7B) substantially reduces the fraction of tetraploid cells
(12.8%) and increases the total fraction of cells in S-phase from 41.2 (untreated) to 56.7% (catalase treated). Similar changes were seen
after treatment with NAC (data not shown). While the exact signaling
relationships responsible for these events is not clear, this suggests
the possibility that antioxidants impair progression into
G2-M.
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Melanoma cells release endogenously generated reactive oxygen
species.
Inhibition of constitutive NF-B activation in malignant cells by
antioxidants suggests that neoplastic cells may be exposed to constant
redox stress that in turn activates the I
B kinase complex. One
potential source of oxidant stress might be reactive oxygen species
produced endogenously by the tumor cell. M1619 cells progressively
reduced ferricytochrome c placed in the buffer medium.
Approximately half the ferricytochrome c reduction was inhibitable by SOD, indicating that O
produced much
of the observed reduction (
A550 after 3 h = 0.012 ± 0.001 with ferricytochrome c alone vs.
0.005 ± 0.001 for ferricytochrome c + SOD;
A550 after 12 h = 0.021 ± 0.001 with
ferricytochrome c alone vs. 0.007 ± 0.001 for
ferricytochrome c + SOD; n = 12 each
and P < 0.001 for both time points). When they were
incubated in buffer alone without serum stimulation, melanoma cells
produced 4.69 ± 0.16 µmol O
/mg cell protein
over 24 h. These results suggest that the source of
oxidant stress driving constitutive activation of NF-
B in these
cells is endogenously generated in an autocrine fashion and is not the
consequence of stimulation by growth factors present in serum.
Endogenous sources of redox stress are supported by the finding that
M1619 cells incubated in serum-free medium for 24 h still
exhibited substantial constitutive NF-
B DNA binding activity in
nuclear protein (Fig. 1E, lanes 3 and
4).
Reactive oxygen species in melanoma cells are generated by an
NAD(P)H-dependent enzymatic activity similar to NQO.
M1619 cells had no measureable xanthine oxidase activity, and no
evidence was detected of mRNA specific for the p22 and
gp91phox components of neutrophil NADPH oxidase
or for the newly described mox-1 (or NOH-1L) oxidase.
Neither cellular reduction of ferricytochrome c nor
proliferation were reduced by the xanthine oxidase inhibitor allopurinol, the cycloxygenase inhibitor indomethacin, the cytochrome P-450 inhibitor cimetidine, the nitric oxide synthase
inhibitor LNAME, or the mitochrondrial respiratory chain inhibitor
rotenone. However, ferricytochrome c reduction was
significantly decreased by the quinone analog capsaicin (Fig.
8A). Capsaicin, as well as the
NAD(P)H oxidase inhibitors diphenylene iodonium chloride and apocynin
(4'-hydroxy-3'-methoxy-acetophenone), significantly reduced
proliferation of M1619 melanoma cells at 48 h (Fig.
8B). Also, apocynin treatment of cells for 24 h reduced
constitutive nuclear translocation of NF-B as assessed by
immunohistochemistry (Fig. 2, C vs. A). Taken
together, these results suggest that the source of endogenous
O
generation stimulating NF-
B activation and
influencing cellular proliferation in this cell line is an NAD(P)H
oxidoreductase activity distinct from the
gp91phox neutrophil NADPH oxidase,
mox-1 or the NOH-1L oxidase.
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DISCUSSION |
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The transcription factor NF-B is increasingly recognized to
play an important role in development and progression of a variety of
malignancies. While the exact signaling relationships linking activation of NF-
B to neoplastic cell proliferation are incompletely understood, NF-
B is constitutively activated in a number of
malignancies (5, 7, 9, 12, 21, 28, 53, 56, 62-64),
and inhibition of NF-
B activation both reduces tumor cell growth
(5, 7, 28, 56) and sensitizes malignant cells to
TNF-
-mediated apoptosis (9, 62, 63). In
addition, NF-
B activation by anti-tumor chemotherapy may play a role
in mediating resistance of tumor cell lines to currently available
chemotherapy treatments by upregulating anti-apoptotic gene
products (9, 62, 63). Thus unraveling the mechanism by
which NF-
B is constitutively activated in neoplasms would represent
an important advance in tumor cell biophysiology. In normal cells,
agonist stimulation activates I
B kinase, leading to I
B
phosphorylation (3, 57), ubiquitination, and proteolytic degradation (46). Uncoupled from its inhibitor, the
NF-
B complex translocates to the nucleus to transcriptionally
regulate expression or repression of target genes (10). In
cultured melanoma cells, constitutive activation of NF-
B is the
result of elevated constitutive activity of the I
B kinase complex
(19), resulting in enhanced degradation of I
B
(53). Thus constitutive activation of NF-
B in tumors
may follow intermediate signaling events that are identical to those
occurring during activation of NF-
B in normal cell lines (3,
57).
The initial upstream response to agonist activation of IB kinase and
subsequent degradation of I
B
in normal cells has been proposed to
be production of O
followed by generation of
H2O2 (51). The compelling argument
for this sequence of events is the observation that many of the known
inducers of NF-
B also induce generation of reactive oxygen species
(2, 3, 51, 52). Human tumor cells produce substantial
amounts of reactive oxygen species spontaneously (18, 41,
60). Mitogenic signaling through both Ras (30) and
Rac (31) is mediated by O
production,
and transfection with the O
-generating oxidase
mox-1 transforms normal cells (58). We therefore
proposed that oxidant stress from generation of reactive oxygen species might play a similar role in the constitutive activation of NF-
B in
malignant melanoma cell lines. Cultured M1619 melanoma cells displayed
prominent constitutive nuclear activation of NF-
B, demonstrated by
conspicuous nuclear NF-
B DNA binding activity in EMSAs (Fig.
1A, lane 1) and abundant immunohistochemical staining for
the p65 component of NF-
B in cell nuclei (Fig. 2A). Constitutive activation of NF-
B was greatly decreased by antioxidant treatment of
melanoma cells with the H2O2 scavenger catalase
(Fig. 1C, lanes 4-6, and Fig. 2B), the
sulfhydryl donor N-acetylcysteine (Fig. 1C, lanes
7-9), or the glutathione peroxidase mimetic ebselen (Fig.
1C, lanes 10-12). Reduction in the amount of
phosphorylated I
B
(Fig. 3) by catalase suggests that antioxidant
treatment prevents NF-
B activation by influencing early signaling
pathways regulating activity of the I
B kinase complex. The
interruption of NF-
B functionality by antioxidant treatment was
confirmed by the observation that catalase treatment of M1619 cells
decreases expression of the NF-
B-regulated (29, 32)
cell cycle protein cyclin D1 (Fig. 4) and that ebselen causes a 40%
reduction in protein expression of melanoma growth stimulatory
activity/GRO-
, an NF-
B-regulated autocrine growth factor
important in melanoma cell proliferation (65, 66). The
effectiveness of catalase in reducing constitutive NF-
B activation
in M1585 melanoma cells (Fig. 1E, lane 2) suggests that
oxidant stress may commonly underlie constitutive NF-
B activation in
other malignant melanoma cell lines. Addition of catalase to growth
medium did not induce prominent apoptosis (Fig. 6, C
and E) or necrosis, suggesting that antioxidant treatment
decreased NF-
B activation by selective interruption of
oxidant-mediated signaling events, and not by indiscriminate cellular
toxicity. Even in serum-free medium, melanoma cells progressively reduced ferricytochrome c in an SOD-inhibitable manner and
continued to prominently display nuclear NF-
B DNA binding activity
in EMSAs (Fig. 1E, lane 4). This implies that the source of
oxidant stress stimulating constitutive NF-
B activation in malignant
melanoma cells is endogenous in origin and autocrine in behavior.
Antioxidant treatment with catalase or ebselen also dramatically
reduced growth of M1619 melanoma cells in a dose-dependent fashion,
whether added at the time of initial cell culture (Fig. 5, A
and C) or 24 h later, when cells had firmly attached
and already begun rapid proliferation (Fig. 5D). M1619 cells
treated with catalase demonstrated an increase in the fraction of cells in S-phase and a decrease in the percentage of polyploid cells (Fig.
7), consistent with impairment of progression into the G2-M phase of the cell cycle. Paralleling their activity for inhibiting constitutive NF-B activation, catalase or ebselen also retarded growth of a variety of malignant cell lines, including M1585 melanoma, adenosquamous lung cancer, and carcinomas of the prostate and breast
(Table 1). The importance of oxidant signaling in growth regulation is
firmly established in normal tissues (13, 27, 30, 31, 50,
61). Our findings add to evidence presented by others (16,
18, 40, 41, 58, 60) that reactive oxygen species are also
important growth signals for development and progression of
malignancies. Because NF-
B is the prototypical oxidant-activated
transcription factor, it is tempting to speculate that oxidant stress
selectively promotes malignant cell growth through activation of
NF-
B. However, other transcription factors such as activator
protein-1 are redox regulated (48), and
H2O2 can activate both phosphatidylinositol
3-kinase (49) and the p44 and p42 extracellular
signal-regulated (ERK1 and ERK2) kinases of the mitogen-activated
protein (MAP) kinase superfamily (1), which are thought to
play key roles in the transduction of mitogenic signals to the cell
nucleus. Also, we have recently reported posttranslational redox
regulation for levels of the early response gene product c-Fos
(13). Thus endogenous autocrine oxidant generation might redundantly impact on malignant cellular proliferation at multiple levels of regulation.
The initial form of reactive oxygen produced as a signaling
intermediate by melanoma cells appears to be O. However, the effectiveness of catalase, but not SOD, at interrupting NF-
B activation (Fig. 1) and malignant cellular proliferation (Fig.
5) suggests that H2O2 formed by dismutation of
O
either spontaneously or by extracellular SOD is
the proximately important oxidant signaling intermediate. The
importance of H2O2 as the relevant signaling
intermediate for oxidant regulation of cell growth has been previously
observed (13, 30, 31, 60, 61). The reactive oxygen species
driving NF-
B activation in M1619 cells are not produced by commonly
considered sources of oxidant generation in cells. Neither cellular
reduction of ferricytochrome c nor proliferation were
reduced by the xanthine oxidase inhibitor allopurinol, the
cycloxygenase inhibitor indomethacin, the cytochrome P-450
inhibitor cimetidine, the nitric oxide synthase inhibitor LNAME, or the
mitochondrial respiratory chain quinone oxidoreductase (complex I)
inhibitor rotenone, suggesting that oxidants important in signaling
transcription and growth were not generated by these oxidases or
mitochondrial aerobic metabolism. We (13) and others
(26, 30, 31, 50, 61) have previously demonstrated the
importance of a putative NADH/NADPH oxidase as a source of signaling
oxidants in normal cells. However, in our M1619 melanoma cell line, we
could not detect mRNA specific for the p22 and
gp91phox components of neutrophil NADPH oxidase
or for the newly described mox-1 (58) or NOH-1L
(4) oxidase.
Our experiments demonstrate the novel finding that reactive oxygen
species driving constitutive activation of NF-B in M1619 melanoma
cells may be generated by a redox couple involving NQO (EC 1.6.99.2),
membrane ubiquinone, and molecular oxygen, with NQO-mediated reduction
of ubiquinone to ubiquinol, which can redox cycle with molecular oxygen
to produce O
. NQO is homodimeric ubiquitous
cytosolic and a membrane flavoprotein that catalyzes the two-electron
reduction of quinones to quinols, which can spontaneously produce a
one-electron reduction of molecular oxygen to O
(23). NQO has been shown to redox couple with and reduce
membrane ubiquinone (35), and both quinone reductase
activity and ubiquinone have been previously established as
requirements for function of the plasma membrane electron transport system (59). This oxidoreductase electron transport system
is an ancient respiratory couple found in all eukaryotic cells. It functions as another system, in addition to utilization of NADH by
mitochrondria and by the pyruvate/lactate couple, for reoxidation of
NADH derived from cellular metabolism to maintain the required NAD+/NADH ratio to support cell viability
(37). In human Namalwa
o cells, which lack
mitochrondria, activity of this plasma membrane oxidoreductase system
is greatly upregulated to support cell growth (37). NQO
differs from other quinone reductases in the cell in that it uses both
NADH and NADPH as electron donors and is selectively inhibited by low
concentrations of dicumarol (22). NQO is abundantly
expressed in tumor cells (Fig. 9). The importance of NQO in producing
growth-signaling reactive oxygen species in M1619 cells is suggested by
experiments demonstrating that dicumarol significantly decreases
ferricytochrome c reduction (Fig. 10A), constitutive activation of NF-
B (Fig. 10, B and
C, and 2D), functionality of NF-
B (reduction
of GRO-
and IL-8 expression, Fig. 10, D and E), and tumor cell proliferation (Fig. 10F) in
this malignant cell line. The importance of endogenous membrane
quinones such as ubiquinone in oxidant generation is indicated by
experiments in which ferricytochrome c reduction was
significantly decreased (Fig. 8A) by the quinone analog
capsaicin (41). Finally, capsaicin, as well as the NAD(P)H oxidase inhibitors diphenylene iodonium chloride and apocynin significantly reduced proliferation of M1619 melanoma cells (Fig. 8B) at concentrations previously reported to block NAD(P)H
flavoprotein-dependent oxidase activity in cultured cells (13,
16, 40, 41). This suggests that the source of endogenous
O
generation for this cell line is a flavoprotein
that utilizes NADPH or NADH as a source of electrons. Like other
flavoenzymes, NQO is inhibited by diphenylene iodonium
(45). Thus our experimental results point to an NQO- and
ubiquinone-dependent electron transport system as the likely source of
growth signaling reactive oxygen species in melanoma and perhaps other
malignant cell lines (Table 1).
An NAD(P)H oxidase system inhibited by capsaicin has been previously
described in malignant cells (41) and in sera from cancer
patients (7, 40). This system, which depends on NQO and
ubiquinone, is also postulated to include a multifunctional hydroquinone oxidase with protein disulfide-thiol interchange activity
associated with the external plasma membrane (42, 43). It
is presently unclear whether O is generated directly
by reduction of molecular oxygen by ubiquinol or is intermediately generated from ubiquinol by an action of this putative multifunctional hydroquinone oxidase. Regardless of which mechanism is ultimately proven, NQO may play an important role in transferring electrons from
NAD(P)H to generate reactive oxygen species important in cell
signaling. Not only does dicumarol inhibit constitutive activation of
NF-
B in tumor cells, but it has also recently been shown to block
the stress-activated protein kinase stress-signaling cascade and
NF-
B pathway in human embryonic kidney 293 cells (17), suggesting that the NQO-ubiquinone couple may represent the common source of proximate messenger reactive oxygen species stimulated by
many of the known inducers of NF-
B (2, 3, 51, 52). Inhibition of oxidant signaling by dicumarol is not from interference with vitamin K epoxide oxidoreductase or a previously unrecognized aspect of vitamin K metabolism, since addition of equimolar
concentrations of vitamin K to growth medium did not impair the
growth-inhibiting effect of dicumarol (Fig. 10F) and does
not reduce the ability of dicumarol to block stress-activated protein
kinases (17). The growth inhibitory effect of dicumarol
may also be relatively specific for tumor cells, since it failed to
significantly reduce proliferation of normal human airway myocytes,
another cell line for which we have previously found reactive oxygen
species important as growth-signaling intermediates (13).
Gene expression of NQO1, the common form of NQO, is greatly
upregulated in tumors of the liver, lung, colon, and breast, compared
with normal tissues of the same origin (11), perhaps to
accommodate the needs of rapidly metabolizing cells to regenerate
NAD+, and polymorphism of NQO1 has been
recently confirmed as a risk factor in lung cancer (14,
38). Thus the redox couple between NQO and ubiquinone may be
relatively more important as a source of growth-signaling reactive
oxygen species in transformed neoplastic cells than in normally
regulated nonmalignant tissues. Confirmation of the importance of NQO
as a source of reactive oxygen species for mitogenic signaling in tumor
cells will require additional studies, including investigation of
NF-
B activation and proliferation in cells after deletion of NQO
activity by a molecular approach.
The findings in this study have two implications. First, others have
suggested that the host environment is the origin of oxidant stress
activating NF-B in tumor cells (20). Our results indicate that reactive oxygen species produced endogenously by this
primordial electron transport system of tumor cells may also activate
NF-
B, and raise the possibility that tumor-derived oxidants could
also have other autocrine functions in maintaining stimulation of other
oxidant-sensitive growth signaling pathways (1, 13, 30, 31, 48,
49), including activation of Jun (17). As a
consequence, fully understanding the origin and metabolic effects of
endogenous reactive oxygen intermediates represents an important new
area of investigation in tumor cell biophysiology. Second, the
significant reduction in tumor growth produced by antioxidants and
quinone reductase inhibitors in the cell lines we studied (Fig. 5 and
Table 1) illustrates the therapeutic potential of certain antioxidant
strategies, either as an adjunctive measure to decrease tumor
resistance to conventional chemotherapy (62, 63) or as a
new stand-alone approach to alter the relationship between host and
parasite, slowing melanoma, prostate, breast, or lung cancer growth in
circumstances where a cure with more radical measures is not possible.
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ACKNOWLEDGEMENTS |
---|
We thank Jeanene Swiggett for assistance with DNA cell cycle analysis, Dr. Helen Gruber for help with photography of immunohistochemical stains, and Dr. James Martin, whose vision, encouragement, and support has made our work possible.
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FOOTNOTES |
---|
This work was funded by grants from the Charlotte-Mecklenberg Health Care Foundation and in part by National Heart, Lung, and Blood Institute Grants HL-40665 (to J. R. Hoidal) and HL-61377 (to A. R. Whorton).
Address for reprint requests and other correspondence: T. Kennedy, Carolinas Medical Center, 410 Cannon Research Center, PO Box 32861, Charlotte, NC 28232 (E-mail: tkennedy{at}carolinas.org).
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.
Received 25 April 2000; accepted in final form 13 October 2000.
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