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Tumor Biology |
Surgical Metabolic Research Laboratory at Lundberg Laboratory for Cancer Research, Department of Surgery, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSStatisticsRESULTSDISCUSSIONREFERENCES |
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-nitro-L-arginine methyl
ester and N-nitro-L-arginine)
reduced tumor growth in vivo by 
50% in both tumor
models. Tumor growth reduction, related to NOS inhibition, was
unrelated to prostanoid production and was an in vivo
phenomenon in both tumor models. Specific inhibitors of inducible NOS
activity, unexpectedly, had no effect in any tumor model, although
inducible NOS protein was present in tumor tissues in large
amounts. A combination of Cox and NOS inhibitors had no additive
effect on tumor growth (MCG 101). Cox inhibition increased tumor tissue
(MCG 101) expression of cNOS mRNA but had no significant effect
on tumor tissue expression of the transferrin receptor, vascular
endothelial growth factor, or basic fibroblast growth factor. NOS
inhibition increased tumor tissue content of cNOS mRNA but
showed as well a trend to increase mRNA content of the transferrin
receptor and vascular endothelial growth factor. Our results suggest
that NOS inhibitors can decrease the local growth of tumors that are
either responsive or unresponsive to Cox inhibition. This effect may
reflect cross-talk between Cox and NOS pathways within or among tumor
cells, or it may represent unrelated effects on tumor and host cells.
Whether NO inhibition may be used therapeutically in clinical tumors
that are unresponsive to eicosanoid intervention remains to be
evaluated. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSStatisticsRESULTSDISCUSSIONREFERENCES |
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,
IL-1ß (7)
, tumor necrosis factor- (8)
,
and
IL-6.4
Thus, the improvement of cachexia and depression of tumor growth by Cox
inhibition (Cox-1 and Cox-2) may be related to reduced production of
prostanoids in either tumor cells or host tissues, or a combination of
both. NOS activities are also related to tumor growth, cellular differentiation, tumor blood flow, and metastatic formation under similar conditions in which Cox inhibitors may act (9, 10, 11, 12, 13) . Interestingly, it has been recognized that production of NO is related to Cox metabolism both in vitro and in vivo during a variety of experimental conditions (14, 15, 16) . Therefore, it is possible that both NO and Cox activities are either alternative or common pathways in the regulation of fundamental processes that determine local and systemic progression of cancer (17) . The aim of the present study was, therefore, to evaluate to what extent Cox and NO-forming pathways may interact in their influence on tumor growth and subsequent development of cancer cachexia. For this purpose, we have used two different mouse tumor models with and without cancer cachexia related to high and low prostanoid production, respectively.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSStatisticsRESULTSDISCUSSIONREFERENCES |
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All animals were housed in groups of five in plastic cages in a temperature-controlled room (24°C) at increased humidity with a 12-h light/dark cycle and were provided free access to standard chow. Tumor tissue or cultured cell suspensions were inoculated under light i.p. anesthesia (100 µg/g ketamin and 5 µg/g xylazin). C57Bl mice were implanted s.c. with 3 mm3 of MCG tumor tissue on both sides of the back to allow unrestrained movement of the animals. The K1735-M2 cell line was suspended in McCoy’s 5A medium in a concentration of 500,000 cells/ml, of which 0.2 ml containing 100,000 cells was inoculated bilaterally in the flanks of the C3H/HeN animals.
Mice were killed by cervical dislocation either 10 days (MCG) or 30 days (K1735-M2) after tumor implantation. The tumors were dissected free for compositional measurements. Food intake, body composition, and tumor dry weight were measured as described elsewhere (22 , 23) .
Provision of Drugs
Most drugs were provided in the drinking water to minimize the
stress reaction by daily injections. Calculation of the dosage of each
drug administered was based on measured water consumption corresponding
to 3 ml/animal and 24 h (20)
. Otherwise, a Cox-2
inhibitor (L-745,337) was given as s.c. daily injections in saline,
according to recommendation of the provider (24)
. Two
hundred and fifty mice were randomly divided into 16 groups, which
received the following treatments with standardized doses.
Experiments in MCG-bearing Mice
For NOS inhibition, the groups were divided as follows: group 1,
L-Name (100 mg/kg/day; n = 17);
group 2, L-NoArg (100 mg/kg/day;
n = 12); group 3;
L-Name plus L-NoArg (100
mg/kg/day of each; n = 9); and group 4,
D-Name (100 mg/kg/day; n = 6). For iNOS inhibition, the groups were divided as follows:
group 5, Amg (100 mg/kg/day; n = 17); and
group 6, Smt (50 mg/kg/day; n = 8). For Cox
inhibition, there was only one group: group 7, indomethacin sodium
(Confortid; 1 mg/kg/day; n = 22), which was
regarded as an unselective Cox-1/Cox-2 inhibitor. For Cox-2 inhibition,
there was only one group: group 8, L-745,337 (10 mg/kg/day;
n = 26). For the combination of Cox and NOS
inhibitors, the groups were divided as follows: group 9, indomethacin
combined with L-Name (1 mg/kg/day plus 100
mg/kg/day; n = 7); and group 10, indomethacin
combined with L-NoArg (1 mg/kg/day plus 100
mg/kg/day; n = 12). There was one control
group: group 11 (n = 54). These tumor-bearing
mice received normal drinking water only.
Experiments in K1735-M2-bearing Mice
For NOS inhibition, there was only one group: group 12,
L-NoArg (n = 10). For iNOS
inhibition, the groups were divided as follows: group 13, Amg
(n = 10); and group 14, Smt
(n = 10). For Cox inhibition, there was only
one group: group 15, indomethacin (n = 10).
There was one control group: group 16 (n = 20). These tumor-bearing mice received normal drinking water only.
L-Name, D-Name, Amg, and L-NoArg substances were from Sigma Chemical Co. (St. Louis, MO). Smt was from Fluka Chemika (Buchs, Switzerland). Indomethacin (Confortid) was from Dumex A/S (Copenhagen, Denmark), provided at effective doses as confirmed by Gelin et al. (20) . L-745,337 was a kind gift from Merck Frosst, Canada Inc. The suggestions of effective in vivo doses of NOS inhibitors were based on published reports (25 , 26) .
Cell Cultures
Tumor cells were grown and harvested for analyses as described
earlier (1)
.
Immune Histochemistry
Tumors were removed and fixed in 4% paraformaldehyde in PBS for
3 days, rinsed, and embedded in paraffin. Tissue sections of 3–4 µm
were cut. After deparaffination, rehydration, and rinsing, sections
were trypsinated for 15 min at 37°C in 0.04% trypsin (Sigma T-8253),
0.01% CaCl2 in Tris-buffered saline (TBS). After
blocking of endogenous biotin activity (Avidin/Biotin blocking kit;
Vector), sections were incubated for 30 min in 5% lipid-free dry milk
in TBS. For NOS detection, (endothelial) sections were incubated 30 min
in normal goat IgG (Santa Cruz Biotechnology) and diluted 1:500
in dry milk/TBS, and Cox sections were incubated 30 min in normal
rabbit IgG (Santa Cruz Biotechnology) diluted 1:500. Test sections were
incubated with antibodies against cNOS (endothelial isoform) diluted
1:200, iNOS diluted 1:200 (Santa Cruz Biotechnology), Cox-1 diluted
1:200, and Cox-2 diluted 1:200 (Santa Cruz) overnight at 4°C. Control
sections (NOS) were incubated in normal rabbit IgG, diluted 1:500, and
control sections (Cox) were incubated in normal goat IgG, diluted 1:500
or with neutralized antibodies overnight. For neutralization,
antibodies were reacted with a 10-fold (by weight) excess of peptide
antigens in 5% dry milk in TBS, 2 h at 37°C. Cox-1 peptide and
Cox-2 peptide were from Santa Cruz Biotechnology. Sections were exposed
30 min at room temperature to biotinylated second antibodies: NOS,
antirabbit diluted 1:200 (Santa Cruz Biotechnology); Cox, antigoat
diluted 1:500 (Dakopatts), followed by 30 min incubation in alkaline
phosphate-conjugated streptavidin diluted 1:150 (Amersham). Fast Red
(Dakopatts) was used as substrate, and counterstaining was done in
Mayers hematoxylin. Mount-Quick Aqueous (Daido Sangyo Co, Ltd,
Japan) was used for mounting.
Western Blot
Tumor tissue was rinsed from debris in 4°C normal saline and
was placed in plastic test tubes and snap frozen in liquid nitrogen.
Specimens were thereafter stored at -84°C until analyses. Tissues
were homogenized by a glass pestle in 4 volumes of PE buffer (10
mM potassium phosphate buffer, pH 6.8, and 1 mM
EDTA) containing 10 mM
3-[(3-cholamidopropyl)dimethyl-ammonio]1-propanesulfonate (Sigma
Chemical Co.), 0.15 µM aprotinin, 2.0 µM
leupeptin, 4.0 µM Pefabloc, and 15 µM
pepstatin (all from Boehringer Mannheim, Mannheim, Germany; Ref.
27
). The homogenate was centrifuged (10,000 x g for 10 min) after determination of protein
concentration in the supernatant (28)
. The samples were
stored at -84°C until analysis. Total protein-equivalents (50
mg/each sample) were diluted in SDS sample buffer and were separated on
7.5% SDS polyacrylamide gel (Bio-Rad Laboratories, Solna, Sweden) in
the Laemmli buffer system (29)
. Proteins were transferred
to a polyvinylidene difluoride membrane (Amersham Laboratories,
Amersham, Buckinghamshire, United Kingdom), which was incubated with
polyclonal rabbit anti-iNOS, or monoclonal mouse anti-eNOS antibody
(diluted 1:1.500 and 1:250, respectively) overnight at 4°C. The
antibodies were purchased from Transduction Laboratories (Lexington,
KY; anti-iNOS and anti-eNOS). Bands were detected by chemiluminescence
using an alkaline phosphatase-conjugated secondary antibody and CSPD
(Western-Light; Tropix, Inc., Bedford, MA; Ref. 30
).
Membranes were exposed to ECL film (Amersham Laboratories).
Tumor Tissue Expression of NOS and Growth Factors
Specimens of viable tumor tissue from a pool of five mice in
each group were used for extraction of total cellular RNA, using
guanidinium isothiocyanate, according to standard procedure
(31)
. Poly(A)+ RNA was selected, and
5–8 µg were either glyoxal treated and separated in 1.2% agarose
gels or run in gels containing formaldehyde and capillary transferred
to Hybond N+ membranes (Amersham). Murine DNA
probes were made by PCR technique using a cDNA library, reversed
transcribed from mRNAs from liver or WBCs. Primers were chosen from Mac
Vectors database Entrez to give fragments of about 350 bp in length
when amplified. These probes were labeled with
[-32P]dCTP in a random priming reaction
using an oligolabeling kit (Roche Diagnostics Scandinavia or
Pharmacia-Biotech). Oligonucleotides of 30–40 bp were also used as
probes. A mix of four murine antisense oligonucleotides/gene were used
to probe VEGF, bFGF, angiogenin, the transferrin receptor, cNOS, and
ß-actin. These were 3'-end labeled using
[-32P]ddATP (Amersham). Prehybridization and
hybridization were performed for 24 h each at 42°C (in a Robbins
oven) in 50% formamide, 5x SSPE, 0.5% SDS, 10x Denhardt’s
solution, and 250 µg/ml denatured low molecular weight DNA, and 50
µg/ml polyadenylic acid. Probe concentration was 5–10 x 105 dpm/ml of hybridization solution.
Postwashes were made in 5x SSPE/0.1% SDS with dilution to 0.1x
SSPE/0.1% SDS, five concentrations in total for 30 min each at 42°C.
Filters were exposed to Hyperfilm MP (Amersham) for 1–11 days.
Densitometric analyses of autoradiograms were performed at 550 nm using
a Shimadzu CS-930 densitometer.
Tumor tissue expression of Cox-2 and ß-actin was measured by semiquantitative PCR. Sixty ng of poly(A)+ RNA from tumor tissue were reversed transcribed using GeneAmp RNA PCR kit (Perkin-Elmer). Oligo d(T)16 was used as a downstream primer. Amplification was done in a reaction volume of 50 µl, using 1:20 volume of the reverse transcriptase-reaction, 2 mM MgCl2, 200 µM deoxynucleotide triphosphates, 0.15 µM of each primer, and 1.25 units of AmpliTaq. HotStart was performed in HotStart 50 tubes (Molecular Bio-Products, Inc., San Diego, CA). Reaction cycles were: denaturing at 94°C for 1 min, annealing at 58°C for 1.2 min, and extension at 72°C for 1 min for 20–36 cycles, followed by a 5-min extension time at 72°C. Aliquots of the PCR reaction (10 µl) were separated in 4% agarose gels and stained in ethidium bromide.
Prostaglandin E2 (125I) Assay System
Blood was collected from tumor-bearing animals on day 10 (MCG)
and day 30 (melanoma) after inoculation. Healthy animals were used as
normal controls. Sodium citrate was used as an anticoagulant, and
indomethacin (10 µg/ml) was used to prevent further breakdown of
arachidonic acid. Specimens were kept on ice and centrifuged at
2500 x g for 10 min at 4°C. After
acidification, ethanol addition, and centrifugation of plasma,
PGE2 was extracted on Amprep C 18 minicolumns
(Amersham RPN 1900), according to the recommendations in the
PGE2 assay system (Amersham RPA 530).
Tumor tissue was homogenized in 0.1 M Tris-HCl buffer (pH 7.4) containing indomethacin as inhibitor. All tubes were kept on ice. After centrifugation, supernatants were treated as mentioned above and applied to C18 minicolumns for extraction of PGE2. In conditioned media from tumor cell cultures, PGE2 was extracted by the same procedure. After conversion of extracted PGE2 by methyl oximation according to the kit instructions, RIA was performed within 6 days. Values were calculated from duplicates.
Fractionation of Eicosanoids
Tissue Preparation.
Tumor tissue from five animals was placed in RPMI 1640 cell culture
medium. A 10% homogenate was prepared in a glass tissue grinder, and 2
ml of the homogenate were incubated with
[3H]arachidonic acid (Amersham; 2 µCi/ml
homogenate) for 3 h at 37°C. Samples were then precipitated with
4 volumes of methanol, vortexed, and refrigerated for 20 min at 4°C.
The samples were centrifuged at 1800 x g.
The supernatant was collected and evaporated under
N2. Finally, the samples were diluted with
methanol to a final concentration of 30% methanol.
Cell Preparation.
Tumor cells were grown in cell culture flasks and harvested 4
days after splitting. A cell suspension containing approximately
3.4 x 107 cells/ml was made. The
cell suspension was homogenized in a glass homogenizer, diluted with 1
volume of McCoy cell culture medium, and incubated with
[3H]arachidonic acid (2 µCi/ml homogenate)
for 3 h at 37°C. The samples were thereafter treated in the same
way as tissue samples.
Tissue and cell extracts (100 µl) were separated on a C18, Pak column (Waters 5 µm; 8-mm inside diameter radius). Eicosanoids were eluted during a series of isocratic elutions with mixtures of methanol and water buffered to pH 5.8, as described (32) .
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Statistics |
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The experimental protocol was approved by the Committee for Animal Ethics at Göteborg University.
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RESULTS |
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35–40% (P < 0.01; Fig. 1
). Reduced tumor growth was related to attenuated anorexia and improved
body composition in MCG-bearing mice (Table 1)
. Indomethacin had no such beneficial effects in melanoma
K1735-M2-bearing mice (results not shown). Thus, melanoma-bearing mice
remained nutritionally unaffected by melanoma growth throughout the
entire experimental period (30 days), despite having a progressively
larger tumor burden than MCG 101-bearing mice close to death. This
discrepancy between the two tumor models may be linked to the fact that
MCG 101 tumors showed a high production of prostanoids (mainly
PGE2, 
1000 ± 100
pg/106 cells/day), whereas the melanoma cells
produced eicosanoids at low amounts (10 ± 5 pg
PGE2/106 cells/day),
evaluated both in vivo and in cultured tumor cells (Fig. 2)
.
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. This difference may indicate that eicosanoids produced by host
cells were also involved.
At the time of sacrifice, MCG 101-bearing mice had elevated plasma
concentrations of PGE2 in the range of
1015 ± 173 pg/ml, which decreased to 90 ± 15 pg/ml by indomethacin treatment and to 234 ± 39pg/ml by L-744,337. Freely fed non-tumor-bearing control mice had
plasma levels of 81 ± 24 pg/ml. The
PGE2 concentrations in tumor tissue were
1000 ± 150 ng/g in MCG 101 tumors 10 days after
tumor implantation in untreated mice. This concentration decreased <20
ng/g by indomethacin treatment, which was comparable with
concentrations of PGE2 in untreated malignant
melanoma-bearing mice (10 ± 2 ng/g tumor tissue) having
plasma concentration of PGE2 (<50 pg/ml) close
to concentrations in healthy non-tumor-bearing mice. Provision of a
Cox-2 inhibitor (L-745,337) decreased PGE2
concentrations in MCG 101 tumors from 840 ± 57 to
180 ± 60 ng/g (P < 0.01).
NOS Inhibition.
Inhibitors (L-NoArg and L-Name) of NO
production decreased tumor growth in vivo by 50% in both
MCG 101 (Fig. 1)
and melanoma-bearing mice (P < 0.01; Fig. 3
). L-NoArg and L-Name
reduced local tumor growth similarly at concentrations used (100 mg/kg
body weight). The stereoisomer D-Name had no
effect on tumor growth, demonstrating NOS specificity by
L-NoArg and L-Name. NOS
inhibition of tumor growth in MCG 101-bearing mice was also associated
with improved carcass fat-free dry weight, whereas the cumulative food
intake was not significantly influenced (Table 1)
. Selective inhibitors
(aminoguanidine and S-methylisothiourea) of iNOS had no
beneficial effect on either tumor growth or nutritional state in any of
the tumor models. The combination of Cox and NOS inhibition did not
show any synergistic effect on tumor growth, food intake, or
nutritional state in any of the tumor models. NOS inhibition
(L-NoArg) had no effect on
PGE2 content in MCG 101 tumor tissue, whereas the
host plasma PGE2 concentrations decreased
significantly from 3000 ± 600 pg/ml to 1521 ± 425 (P < 0.05). NOS inhibition by
L-Name (1 mM) reduced only
marginally proliferation of cultured MCG 101 (6 ± 2%;
P < 0.05) and melanoma cells (7 ± 3%; P < 0.05).
|
. Cox-2 was expressed 500-fold higher in MCG tissue
compared with melanoma tissues evaluated by quantitative PCR (Fig. 5)
. Tumor tissue content of Cox-1 and Cox-2 protein was not changed by
indomethacin treatment evaluated in MCG 101-bearing mice (not shown).
Both tumors (MCG 101 and K1735-M2) contained substantial amounts of
both cNOS and iNOS protein, with even distribution throughout the tumor
tissue (Fig. 4B)
. Particularly, melanoma tumors seemed to
have high levels of iNOS, which was confirmed by Western blot analysis
(Fig. 6)
.
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. Provision of L-NoArg to MCG 101-bearing mice also
increased tumor cNOS mRNA. It also showed a trend to increased tumor
expression of the transferrin receptor and VEGF, but
L-NoArg provision was without effect on the expression of
angiogenin and bFGF. The combination of indomethacin and
L-NoArg had no synergistic effect on eNOS mRNA expression
in tumor tissue (Table 2)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSStatisticsRESULTSDISCUSSIONREFERENCES |
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A number of recent reports have indicated that NOS activities may
either stimulate or retard tumor growth by effects that are
superficially similar to mechanisms exerted by Cox pathways (10
, 40)
. Such observations are interesting because NO mediates
(14)
, or is at least related to, Cox activities and tumor
growth (10
, 41) . Therefore, we decided to evaluate whether
provision of NOS inhibitors at nontoxic doses attenuates cancer
cachexia and tumor growth in addition to Cox inhibition (6
, 14)
. For this purpose, we used two different tumor models: one
model with cachexia being significantly dependent on Coxs, and another
model without cachexia, being unresponsive to Cox activity for tumor
growth. Our results demonstrate that NOS inhibition by oral intake of
arginine analogues (L-Name and L-NoArg)
inhibited tumor growth in vivo by 50% in both tumor
models independently of eicosanoid formation, whereas cultured tumor
cells were only marginally affected by NOS inhibition (<10%). These
findings agree with conclusions on the role of NOS promotion for tumor
growth in experimental and clinical cancer (21)
. However,
specific inhibitors of iNOS activity had no effect in any of our tumor
models, although iNOS protein was present in both kind of tumors. This
phenomenon may indicate a poor correlation between the cellular content
of iNOS and NO formation, as reported recently for arachidonic
acid-metabolizing enzymes and generation of corresponding metabolites
(39)
. The literature on NO formation and tumor growth is
not unanimous (42
, 43)
. Some studies report that low
levels of NO promote tumor growth, whereas high levels may be
inhibitory. Also, it is not clear whether stimulation of NO production
by iNOS induction in tumor cells may promote tumor cell proliferation
(44)
, whereas iNOS induction in surrounding host cells may
prevent local tumor growth (45)
. Dong et al.
(13)
have reported that iNOS expression in K1735 melanoma
cells was inversely related to metastatic growth and progression,
findings which agreed with the observation that various clones of
melanoma cells expressed iNOS above or below detection levels in
tissues (46)
. Therefore, it is not possible to judge why
our tumors, with detectable iNOS, did not respond significantly to iNOS
inhibitors. Possible explanations may be insufficient drug
concentrations after oral provision, or that iNOS is differentially
important for local tumor growth and metastatic spread
(39)
. With this uncertainty in mind, our findings suggest
a model where downstream pathways for Cox and NO activation end up into
a common effector mechanism(s) for MCG tumors, whereas other mechanisms
may be present in K1735-M2 melanomas. These results both agree and
disagree with models suggested by others for normal cellular
physiology, where integrated functions between Cox and NOS production
seem to be a general phenomenon (47, 48, 49)
.
Previous and present results suggest that the beneficial effects of indomethacin and NO inhibition may in part be host related. This conclusion is based on the observation that in vivo effects were more pronounced than in vitro effects, and that NOS inhibition in MCG-bearing mice did not decrease tumor content of prostaglandins but reduced circulating plasma PGE2, which was associated with improvement in nutritional state (fat-free carcass weight). These observations suggest interaction between Cox and NOS pathways among host tissues and not only within tumor tissue (50, 51, 52) . Alternatively, tumor growth inhibition by NO may not be independent of prostanoid pathways (53) . However, if completely separate pathways occurred, one should expect additive effects by Cox and NOS inhibitors in MCG tumors, which was not observed.
We have earlier reported that provision of indomethacin to MCG
101-bearing mice increased tumor expression of various growth factors
(IL-6, tumor necrosis factor-, granulocyte-macrophage
colony-stimulating factor, and transforming growth factor
ß3) and decreased expression of bFGF and
angiogenin, but it left the tumor expression of VEGF, epidermal growth
factor, platelet-derived growth factor-A, platelet-derived growth
factor-B, IL-1, and the transferrin receptor unchanged
(1)
. The present study adds to previous information that
transcriptional expression of cNOS mRNA in tumor tissue was increased
2-fold by either indomethacin or L-NoArg treatment (Table 2)
, confirmed by Western blot analyses.4 Thus,
up-regulation of mRNAs in tumor tissue suggests that both Cox and NOS
pathways are related to cellular growth factors in either tumor or in
host (endothelial) cells. A decreased flow of prostanoid metabolites
and of NO after provision of inhibitors up-regulated cNOS expression by
negative feed back to support continuous tumor growth
(54)
. These findings illustrate a phenomenon of cross-talk
between Cox and NOS pathways (48)
. Although the model is
not yet defined, our observations may include loops with positive and
negative feedback for genes that may in the future be possible to use
therapeutically. For example, cancer cells with mutated p53 had
accelerated tumor growth associated with increased expression of VEGF
for neovascularization related to hypoxia and subsequently assumed
increased NO formation (11
, 55
, 56)
. It is generally
anticipated that both indomethacin and NO influence the tumor
vasculature, including effects on tumor blood flow, endothelial cells,
and tumor nutrient uptake. However, unpublished work in our MCG
101-bearing mice has, thus far, not confirmed that Cox inhibition
reduced angiogenesis in MCG 101 tumors. It now remains to be evaluated
whether clinical tumors are sensitive to NOS inhibition in a way that
may add to our treatment reported previously of cancer patients with
Cox inhibitors alone (indomethacin), particularly in patients with
tumors unresponsive to Cox intervention (6
, 57)
.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in parts by Grant 2014-B98-12XAC from
the Swedish Cancer Society, Grants K99-72X-08712-11A and
K99-73X-11611-04A from the Medical Research Council, and grants from
the Tore Nilson Foundation, Assar Gabrielsson Foundation (AB Volvo),
Jubileumskliniken Foundation, IngaBritt and Arne Lundberg Research
Foundation, Swedish and Göteborg Medical Societies, the Medical
Faculty, Göteborg University, and the Gunnar Nilsson Memorial
Foundation. 
2 To whom requests for reprints should be
addressed, at Department of Surgery, Sahlgrenska University Hospital,
S-413 45 Göteborg, Sweden.
3 The abbreviations used are: Cox, cyclooxygenase;
IL, interleukin; NOS, nitric oxide synthase; cNOS, constitutive
NOS; iNOS, inducible NOS; PGE2, prostaglandin
E2; L-Name,
N-nitro-L-arginine methyl
ester; D-Name,
N-nitro-D-arginine methyl
ester; L-NoArg,
N-nitro-L-arginine; Amg,
aminoguanidine hemisulfate; Smt, S-methylisothiourea
sulfate; L-745,337, 5-methane
sulfonamido-6-(2,4-difluoro-thiophenyl)-1-indanone; VEGF, vascular
endothelial growth factor; bFGF, basic fibroblast growth factor.
Received 7/19/99. Revised 12/16/99. Accepted 1/19/00.
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REFERENCES |
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)
and with 
indomethacin treatment before sacrifice, as described in
"Materials and Methods." B, eicosanoids synthesized
in cultured MCG 101 cells compared with the synthesis in K1735-M2
cells. For details, see "Materials and Methods." Eicosanoids were
separated as described 
