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*
UCLA-Wadsworth Pulmonary Laboratory and
Jonsson Comprehensive Cancer Center, University of California, Los Angeles, School of Medicine and West Los Angeles Veterans Affairs Medical Center, Los Angeles, CA 90073;
G. D. Searle, St. Louis, MO 63167; and
§
La Jolla Institute of Allergy and Immunology, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The initiation of prostanoid synthesis from arachidonic acid involves the enzyme referred to as COX, which has also been termed PGH synthase or PG endoperoxide synthase (22). Two isoenzymes have been identified: a constitutive form (COX-1) and an inducible isoenzyme (COX-2) (23, 24, 25). COX-2 is up-regulated in response to a variety of stimuli, including growth factors and cytokines (23). Because it can lead to enhanced PGE2 production and subsequent cytokine imbalance in vivo, tumor expression of COX-2 may be instrumental in the generation of tumor-induced abrogation of T cell-mediated antitumor responses (5). COX-2 has been implicated in the development of colon cancer and may play a role in promoting invasion, metastasis, and angiogenesis in established tumors (26, 27, 28, 29). In addition to lung and colon carcinomas (5, 30, 31, 32), COX-2 has recently been reported to be expressed in a variety of human malignancies (33, 34, 35, 36, 37, 38). We report here that specific COX-2 inhibition serves to restore the tumor-induced imbalance in IL-10 and IL-12 and promotes antitumor responses in an immunocompetent murine lung cancer model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Indomethacin and aspirin were obtained from Sigma (St. Louis, MO). NS-398 [N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide], and PGE2 were purchased from Cayman Chemicals (Ann Arbor, MI). SC-58236, anti-PGE2 (2B5 mAb), and isotype-matched control mouse IgG1 (MOPC21) were generously provided by Searle (Skokie, IL). Recombinant mIL-1ß and rat anti-mouse anti-CD40 mAb were obtained from PharMingen (San Diego, CA).
Stable transfection
A 2.3-kb BamHI-XhoI fragment containing the open reading frame for a polypeptide of 604 amino acids of murine COX-2 was isolated and cloned into the BamHI-XhoI site of the eukaryotic expression vector pCR 3.1 (Invitrogen, San Diego, CA). For the antisense insert, a PCR fragment was generated from the sense construct utilizing the T7 promoter as the 5' primer binding site and positions 725–701 of the murine cDNA as the 3' primer binding site. The PCR fragment was cloned into the pCR 3.1 TA vector. In this vector, transcription of the cDNA is controlled by the CMV promoter. This vector also contains the neomycin resistance gene that allows for selection in G418 (Life Technologies, Rockville, MD). COX-2 sense and antisense-oriented expression vectors were prepared. These expression vectors were transfected into the 3LL cell line using the superfect transfection reagents (Qiagen, Los Angeles, CA). 3LL cells were also transfected with the PCR 3.1 control vector that did not contain the COX-2 insert. After transfection, the 3LL cells were selected in 500 µg/ml of G418 for 10 days. After selection, 3LL clones expressing COX-2 sense, antisense, and control vector constructs were isolated by limited dilutions from 96-well plates. 3LL COX-2 sense and antisense clones were initially screened based on PGE2 production. The 3LL COX-2 sense clones produced 7–9 ng/ml/105 cells of PGE2, whereas the COX-2 antisense clones produced in the range of 105–285 pg/ml/105 cells. The clones were further characterized for COX-2 mRNA and protein by Northern and Western blot analysis, respectively. The 3LL COX-2 antisense-transfected clones expressed less COX-2 mRNA and protein than did the 3LL parental tumor cells, 3LL COX-2 sense, or 3LL control vector-transfected clones (data not shown). Northern blot analysis for the COX-1 gene message in the 3LL COX-2 antisense, 3LL COX-2 sense, and 3LL control vector clones showed that the COX-1 message remained unaltered (data not shown).
Northern blot analysis
To determine the time course of COX-1 and COX-2 gene expression in 3LL cells, 3LL COX-2 sense clone (4SC7-3LL), and 3LL control vector clone (CV-3LL) after IL-1ß stimulation, 3LL cells cultures were treated with or without mIL-1ß (150 U/ml) for 1–24 h, and total RNA was isolated. Cell pellets for each of the sample time points were lysed in 4 M guanidine isothiocyanate solution by gently pipetting. A quantity of 0.2 vol of chloroform was added to the cell lysate and kept on ice for 10 min. After centrifugation at 12,000 x g for 10 min at 4°C, the upper aqueous phase was transferred to a new tube. Equal volumes of isopropanol were added to the upper aqueous phase and kept on ice for 45 min. After centrifugation at 12,000 x g for 10 min at 4°C, the RNA pellet was washed twice with 75% ethanol and dried at room temperature. The dried RNA pellet was dissolved in 0.1% diethyl pyrocarbonate-treated double-distilled water and adjusted to a final concentration of 1 µg/ml. Northern blotting analysis was done as previously described to detect COX message (5). Briefly, 10 µg/sample for each of the conditions was electrophoresed through a 10% denaturing formaldehyde agarose gel, and the RNA was then transferred to a Hybond nylon membrane (Amersham, Arlington Heights, IL) in 20x sodium chloride sodium citrate (SSC). The RNA was fixed to the nylon membrane by UV cross-linking (Stratagene, San Diego, CA). Both prehybridization and hybridization were performed at 68°C in rapid hybridization solution obtained from Amersham. Duplicate filters were hybridized overnight with cDNA probes for murine COX-1, COX-2, and ß-actin control. The probes were labeled with a [32P]dCTP using a nick translation kit (BRL, Bethesda, MD). The probes were cleaned by passage through a Pharmacia Sephadex G-50 column (Piscataway, NJ). After hybridization, the filters were washed twice for 15 min at room temperature in 2x SSC/0.1% SDS and once for 10 min at 42°C and three times at 68°C in 2x SSC/0.1% SDS. The filters were exposed to Kodak XAR-5 film overnight at -80°C, and densitometric analysis was performed.
Cell culture
Murine Lewis lung carcinoma cell line (3LL) was obtained from American Type Culture Collection (ATCC; Manassas, VA). The COX-2 antisense 3LL clone (1ASE7-3LL), the COX-2 sense clone (4SC7-3LL), and the control vector-transfected cells (CV-3LL) were utilized for these studies. The cells were routinely cultured as monolayers in 25-cm3 tissue culture flasks containing RPMI 1640 medium (Irvine Scientific, Santa Anna, CA) supplemented with 10% FBS (Gemini Biological Products, Calabasas, CA), penicillin (100 U/ml), streptomycin (0.1 mg/ml), 2 mM glutamine (JRH Biosciences, Lenexa, KS) and maintained at 37°C in a humidified atmosphere containing 5% CO2 in air. The cell line was Mycoplasma free, and cells were utilized up to the 10th passage before thawing frozen stock 3LL cells from liquid N2. For experiments utilizing 3LL, 1ASE7-3LL, 4SC7-3LL, or CV-3LL cell supernatants, 1 x 105 cells/ml were cultured in 6-well plates in RPMI 1640 with or without specific COX inducers or inhibitors. For experiments utilizing aspirin, 105 cells/ml were incubated for 2 h in the presence of aspirin (50 µg/ml). The tumor cells were then washed twice in PBS and replated at 105 cells/ml. After a 24-h culture period, the tumor cell supernatants were collected and PGE2 levels measured by enzyme immunoassay (EIA).
Splenocyte IL-10 production
Splenocytes were isolated from normal mice using a standard protocol (39). Splenocytes were cultured at a concentration of 2 x 106 cells/ml in tumor cell supernatants or in tumor cell supernatants from 3LL cells that were treated with COX inhibitors (aspirin, indomethacin, NS-398, SC-58236, anti-PGE2 mAb, or control Ab). After a 72-h incubation, IL-10 production by splenocytes was assessed by ELISA. Splenic lymphocytes were isolated from mice bearing 1ASE7-3LL tumors, 4SC7-3LL tumors, CV-3LL tumors, SC-5236-treated, indomethacin-treated, and control mice bearing 14-day-old 3LL tumors. Splenic lymphocytes were cultured in control medium (CM) at a concentration of 2 x 106 cells/ml, and IL-10 concentrations were measured after 6 days.
APC isolation
APC were purified from total splenocyte suspension by Ab-mediated complement lysis of T and B cells. The Abs used for these depletions included TIB207 (anti-CD4), TIB150 (anti-CD8), and TIB146 (anti-B lymphocytes). Hybridoma cell lines were purchased from ATCC. After RBC depletion, splenocytes were incubated with a mixture of mAbs and rabbit complement for 60 min at 37°C. After Ab depletion, APCs were washed twice in CM. Staining for cell surface markers with Abs to CD11b, CD11c, and F480 showed that cells were >95% APC following Ab depletion. Less than 5% of cells stained positively for CD3 following Ab depletions. APC (5 x 106 cells/ml) from tumor-bearing mice treated with the COX-2 inhibitor SC-58236, from untreated tumor-bearing controls and control non-tumor-bearing animals were stimulated with anti-CD40 (5 µg/ml). After a 72-h culture, IL-12 levels were determined by ELISA. For macrophage purification, APC suspension was cultured in 6-well plates overnight. After a 24-h culture, non-adherent cells were removed, adherent cells were washed twice with PBS, and macrophages were removed by trypsinization. Macrophages (5 x 106 cells/ml) were stimulated with anti-CD40 (5 µg/ml) in CM, 3LL supernatant, 1ASE7-3LL supernatant, 4SC7-3LL supernatant, CV-3LL supernatant, in media with PGE2, anti-PGE2 mAb, or isotype-matched IgG control Ab, MOPC21 (10 µg/ml).
Mice
Pathogen-free-female C57BL/6 mice (8–12 wk of age) were obtained from Harlan Laboratories (Indianapolis, IN) and maintained in the West Los Angeles Veterans Affairs Animal Research Facility. IL-10 transgenic mice were made by standard methods at University of California, Los Angeles, Transgenic Mouse Core Facility as described previously.
3LL tumor model
For tumorigenesis experiments, 5 x 105 3LL parent, 1ASE7-3LL, 4SC7-3LL, or CV-3LL cells were inoculated on the right suprascapular area in C57BL/6 mice, and tumor volumes and survival were monitored. Tumor growth was assessed three times per week following tumor implantation. Two bisecting diameters of each tumor were measured with calipers. The volume was calculated using the formula (0.4) x (ab2), with a as the larger diameter and b as the smaller diameter. Mice were pretreated with indomethacin (10 mg/ml in drinking water) or SC-58236 (3 mg/kg 3 times per week i.p.) 1 wk before the tumor cell inoculation and for the duration of the experiment. Mice were pretreated with anti-PGE2 mAb or control Ab 24 h before tumor inoculation and three times a week for the duration of the experiment (10 mg/kg i.p.). On day 14 after tumor inoculation, non-necrotic tumors and splenic lymphocytes were isolated from tumor-bearing mice for determination of PGE2 and IL-10 concentrations. PGE2 and IL-10 levels were evaluated in tumor homogenates as described previously (40). Following RBC and macrophage depletion, 6-day splenic lymphocyte culture supernatants were assessed for cytokine production. To compare 3LL, 4SC7-3LL, CV-3LL, and 1ASE7-3LL tumor formation in the lungs of C57BL/6 mice, 5 x 105 tumor cells were inoculated i.v. via a lateral tail vein. After 34 days, mice were sacrificed, and lungs were isolated and perfused by 10% formalin. Following fixation in 10% formalin for 48 h, lungs were embedded in paraffin. Sections (3–4 µm) were prepared for hematoxylin and eosin staining and histopathological examination. Tumor burden in lung sections was evaluated by microscope examination with a calibrated optical grid (a 1-cm2 grid subdivided into 100 1.0-mm2 squares). The total number of positive squares (with tumor occupying >50% of area) was determined for 4–6 separate high-power fields from three histologic sections as described previously (41). To evaluate IL-10-mediated immunosuppression in vivo, the splenic T lymphocytes from C57BL/6 or from IL-10 transgenic mice were isolated from spleens with Dynal beads (Dynal, Great Neck, NY) using the manufacturer’s protocol. A total of 5 x 107 T lymphocytes/mouse was transferred to SC-58236-pretreated C57BL/6 mice 24 h before and 1 wk after 3LL tumor cell inoculation. Tumor volumes were assessed three times per week.
IL-10 and IL-12 ELISA
IL-10 protein concentrations from murine splenocytes cultured in CM or 3LL cell supernatants were determined by IL-10-specific ELISA as described previously (4). Briefly, 96-well Costar (Costar, Cambridge, MA) plates were coated overnight with 4 µg/ml of mouse anti-IL-10 mAb (PharMingen). The wells of the plate were blocked with 10% FBS (Gemini Biological Products) in PBS for 30 min. The plate was then incubated with the Ag for 1 h and excess Ag was washed off with PBS/Tween 20. The plate was incubated with 1 µg/ml of biotinylated mAb to IL-10 from PharMingen for 30 min, and excess Ab was washed off with PBS/Tween 20. The plates were incubated with avidin peroxidase, and after incubation in o-phenylenediamine substrate, the subsequent change in color was read at 490 nm with a Dynatech MR5000 spectrophotometer (Chantilly, VA). The recombinant IL-10 used in the assay as a standard was obtained from PharMingen. The sensitivity of the IL-10 ELISA was 15 pg/ml. For IL-12 measurements, an IL-12 ELISA kit was utilized (BioSource International, Camarillo, CA) and measurements were performed according to the manufacturer’s instructions. The sensitivity of the IL-12 ELISA was 5 pg/ml.
PGE2 EIA
PGE2 concentrations were determined according to the Cayman Chemicals EIA kit protocol as described previously (5). Briefly, 96-well Costar plates were precoated overnight with 4 µg/ml of goat anti-mouse PGE2 (BioSource International). PGE2-acetylcholinesterase conjugate, mouse anti-PGE2 mAb, and either standard or sample were added to each well. After an 18-h incubation at 25°C, the plate was washed five times to remove all unbound reagents. Ellman’s reagent was then added to each well, and absorbance was determined at 405 nm by a Dynatech MR5000 spectrophotometer.
Statistical analysis
All in vitro results are representative of at least three independent experiments performed in triplicate. In vivo experiments were performed with at least six to eight mice per group. Differences between experimental vs control values were evaluated by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lewis lung carcinoma (3LL) is a weakly immunogenic murine lung
cancer that has been shown previously to produce PGs (42).
The two isoforms of COX were evaluated in 3LL. The constitutive enzyme,
COX-1, has been previously found to be present in most cells and
tissues, whereas the inducible isoenzyme, COX-2, is expressed in
response to a variety of stimuli (23). To determine the
time course of COX-1 and COX-2 gene expression,
3LL cells were stimulated with IL-1ß for various durations, and
Northern blot analysis was performed with labeled COX-1 and COX-2 cDNA
probes. As shown in Fig. 1
, the
COX-1 and COX-2 genes are both expressed in 3LL
cells. COX-2 mRNA was induced with maximal induction occurring 2 h
after incubation with IL-1ß. In contrast, COX-1 mRNA expression
remained unchanged.
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Based on previous studies in a variety of host cells and tumors, we anticipated that high-level PG production by 3LL would be COX-2 dependent. Initially, several 3LL COX-2 antisense, sense, and control vector-transfected clones were evaluated for PGE2 production, COX-1 mRNA expression, COX-2 mRNA expression, and protein production as well as tumor growth in vivo. In the initial characterization of the clones, the following results were obtained for which data are not shown. The 3LL COX-2 antisense-transfected clones expressed less COX-2 mRNA and protein than did the 3LL parental tumor cells, COX-2 sense, and control vector-transfected cells. The 3LL COX-2 sense clone expressed higher levels of COX-2 mRNA than the parental or control vector-transfected 3LL cells. In the 3LL COX-2 antisense, COX-2 sense, and control vector clones, COX-1 gene expression remained unaltered. All antisense COX-2 clones evaluated showed diminished tumor growth in vivo, whereas the in vivo growth of CV-3LL and sense clones were indistinguishable from that of the parental tumor.
To further assess the role of 3LL COX-2 expression in modulation of
cytokine production and tumorigenesis, a 3LL COX-2 antisense clone,
1ASE7-3LL, that constitutively produced 90 ± 8
pg/ml/105 cells/24 h was selected for evaluation.
The 3LL COX-2 sense clone 4SC7-3LL produced 7–9 ng/ml/10
5 cells of PGE2 in 24
h was selected for further evaluation. The parental 3LL cell line and
3LL control vector-transfected clones (CV-3LL) constitutively produced
between 2.5 and 3.5 ng/ml/105 cells of
PGE2 in 24 h. Stimulation of parental 3LL
and CV-3LL with mIL-1ß consistently led to a 2- to 3-fold increase in
PGE2 production. mIL-1ß did not augment
PGE2 levels in the sense clone 4SC7-3LL (Fig. 2). In COX-2 antisense transfectants this
induction of PGE2 was abrogated completely as
shown for 1ASE7-3LL in Fig. 2. Pharmacological agents that
differentially block COX isoenzymes also were utilized to determine the
relative contribution of each isoenzyme to high-level
PGE2 production (Fig. 2). The specific COX-2
inhibitors NS-398 and SC-58236, as well as indomethacin and aspirin,
which block both isoenzymes, were utilized. Treatment of 3LL cells with
indomethacin significantly decreased both constitutive and
mIL-1ß-stimulated PGE2 production (Fig. 2).
Aspirin was used to assess abrogation of COX-1. Aspirin has been shown
to inhibit both COX-1 and COX-2 isoenzymes irreversibly by covalent
modification (43). When aspirin is removed from the
medium, however, newly synthesized IL-ß-induced COX-2 is unaffected
and therefore active (44). When 3LL cells were treated
with aspirin for 2 h and then washed, mIL-1ß-stimulated levels
of PGE2 production were maintained in 3LL
supernatants. This suggests that following IL-1ß stimulation,
high-level PGE2 production by 3LL cells is COX-2
dependent. Because specific COX-2 inhibition down-regulated the
baseline, nonstimulated level of PGE2 production
as well as the IL-1ß-induced levels (Fig. 2), we conclude that COX-2
also appears to contribute significantly to constitutive
PGE2 production by 3LL cells. The baseline
constitutive COX-2 mRNA expression and protein production in 3LL
parental cells is consistent with previous studies that have found
serum to induce COX-2 (45, 46). Constitutive expression of
COX-2 in normal host cells and tumor cells also has been described
(47, 48). Thus, treatment of 3LL cells with mIL-1ß
induces COX-2 mRNA expression and this high-level COX-2 expression is
responsible for increased PGE2 levels in 3LL
cells (p < 0.05, Figs. 1
and 2).
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Previous studies indicate that the tumor-bearing state is often
characterized by an increase in IL-10 production but diminished
capacity to produce IL-12 (6, 18). One possibility is that
tumor or host cell-derived PGE2 is the cause of
both augmentation of IL-10 and the simultaneous decrement in IL-12
production (5). To study the effect of 3LL tumor
cell-derived PGE2 on splenocyte production of
IL-10, splenocytes from normal C57BL/6 mice were cultured in 3LL
supernatants. The supernatants were collected after a 24-h incubation
with various combinations of mIL-1ß, COX inhibitors,
anti-PGE2, or control mAb. Splenocyte-derived
IL-10 concentrations were measured after a 72-h incubation of
splenocytes in tumor-conditioned medium. Whereas supernatants
themselves did not contain detectable concentrations of IL-10 (data not
shown), tumor supernatants from 3LL, CV-3LL, and 4SC7-3LL caused an
increase in splenocyte IL-10 production (Fig. 3). Neither COX inhibitors nor IL-1ß
itself had a direct effect on constitutive splenocyte IL-10 production
(data not shown). 3LL COX-2 expression and PGE2
production were predominantly responsible for enhanced splenocyte IL-10
production. This was evidenced by the fact that the increase in
splenocyte-derived IL-10 production was significantly inhibited by
treatment of tumor cells with COX-2 inhibitors, including
indomethacin, NS-398, or SC-58236 (Fig. 3, p < 0.05).
COX-2 antisense-transfected tumor supernatants also had a decreased
capacity to induce IL-10 as shown for 1ASE7-3LL in Fig. 3. The
abrogation of splenocyte IL-10 induction by specific
anti-PGE2 mAb, but not control Ab, confirmed
that PGE2 was the COX metabolite in the tumor
supernatant responsible for IL-10 induction. In experiments that
evaluated the production of splenic lymphocyte and macrophage IL-10 in
response to tumor supernatants, we found that lymphocytes made the
greatest contribution to IL-10 synthesis (data not shown).
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). A similar decrement in
macrophage-derived IL-12 was demonstrated when
PGE2 was added to macrophages in CM. Ab-mediated
neutralization of PGE2 in 3LL supernatant caused
a restoration of macrophage IL-12 production to levels observed when
macrophages were cultured in CM alone. Similarly, macrophage IL-12
production in 1ASE7-3LL supernatant was comparable to levels in CM.
These findings indicate a tumor COX-2-dependent regulation of IL-10 and
IL-12 in host lymphocytes and macrophages. Furthermore,
PGE2 appears to be the COX-2 metabolite
responsible for increased lymphocyte-derived IL-10 and decreased
macrophage IL-12 production in vitro.
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To determine the effect of tumor COX-2 expression on tumorigenesis
in vivo, we studied tumor burden in the lungs of C57BL/6 mice after
i.v. injection of either parental 3LL cells, CV-3LL, 4SC7-3LL, or
1ASE7-3LL transfectants. Compared with mice bearing parental 3LL,
CV-3LL, or 4SC7-3LL, mice injected with 1ASE7-3LL-transfected cells
showed a significant reduction in tumor burden 34 days after tumor
inoculation (Fig. 5A,
p < 0.01). Histologic evaluation of lung sections
revealed that 1ASE7-3LL cells formed discrete tumor nodules that were
extensively infiltrated with lymphocytes (Fig. 5, D and
E). In contrast, parental 3LL, CV-3LL, or 4SC7-3LL injection
led to formation of significantly larger tumor nodules that evidenced
homogeneous tumor fields without inflammatory infiltration (Fig. 5, B and C).
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). Whereas the proliferation of
1ASE7-3LL in vitro was indistinguishable from that of the parental 3LL
line (data not shown), the 1ASE7-3LL showed a significant reduction in
tumorigenesis in vivo that was comparable to mice bearing parental 3LL
tumors receiving COX-2 inhibitors or
anti-PGE2 mAb (Fig. 6). Mice bearing
1ASE7-3LL tumors and those treated with
anti-PGE2 mAb or COX-2 inhibitors had
prolonged survival (median 50 days) compared with control tumor-bearing
mice or mice treated with control Ab (median 30 days, p
< 0.05; data not shown).
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) and splenocytes
(Table II). Tumor nodules from 3LL-,
CV-3LL-, and 4SC7-3LL-bearing mice produced significantly more
PGE2 and IL-10 than did tumor nodules from
anti-PGE2 mAb-, indomethacin-, or
SC-58236-treated mice (Table I). The 1ASE7-3LL tumors also evidenced
reduced PGE2 and IL-10 production within tumor
tissues, and mice bearing these tumors had significant reductions in
IL-10 and PGE2 within the spleen compared with
tumor-bearing controls (p < 0.05, Tables I and
II). Pharmacological inhibition of COX in vivo also reduced IL-10 and
PGE2 production by splenic lymphocytes compared
with untreated tumor-bearing mice (Table II).
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). APC
from both SC-58236-treated and 1ASE7-3LL tumor-bearing mice produced
significantly more IL-12 than APC from untreated
tumor-bearing controls (p <
0.05).
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). In contrast, those mice that received
transfer of equivalent numbers of normal C57BL/6 lymphocytes showed no
modification in the COX-2 inhibitor-mediated antitumor response. Thus,
the antitumor response in this model appears to be due to a COX-2
inhibitor-induced down-regulation of the immunosuppressive effect of
lymphocyte-derived IL-10.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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COX-2 isoenzyme expression is significantly increased in human lung cancers compared with normal, non-cancerous lung tissues (5, 32). Our previous studies documented an immunosuppressive network in human non-small cell lung cancer (NSCLC) that is due to overexpression of tumor-derived COX-2 (5). We report here that 3LL cells express COX-2 at low levels constitutively, and this isoenzyme is induced following stimulation with mIL-1ß in vitro. In contrast, COX-1 mRNA is expressed constitutively and expres- sion remains unchanged following IL-1ß stimulation. The increase in COX-2 mRNA in 3LL cells correlated with increases in COX-2 protein and PGE2 synthesis. High-level PGE2 production by 3LL cells after stimulation with IL-1ß was documented to be COX-2 dependent as evidenced by genetic or pharmacologic inhibition of COX-2 in 3LL cells in vitro. Thus, the capacity for COX-2 induction in 3LL was similar to that previously described in human lung cancer and suggested that these tumor cells would be suitable for assessment in vivo (5).
In vivo inhibition of COX-2 with either indomethacin or SC-58236 led to reduction in tumor growth and prolonged survival. Because drugs that inhibit COX may have effects in addition to COX inhibition (50, 51, 52), we also assessed the involvement of COX-2 expression in tumorigenesis in experiments performed with 3LL cells genetically modified with COX-2 antisense or control constructs. Although the 3LL COX-2 sense or control vector-transfected tumors had growth rates comparable to the parental 3LL cells, the COX-2 antisense-transfected 3LL revealed a significant reduction in tumorigenesis that was comparable to tumors in mice receiving COX-2 inhibitors. Histologic evaluation of regressing tumors following COX-2 inhibition revealed marked lymphocytic infiltration. The histologic appearance suggested that an immune-mediated pathway was operative in mediating tumor reduction. To assess this possibility, we evaluated modulation of cytokine balance that accompanied COX-2 inhibition in vivo.
Previous studies suggest that populations of T cells in the tumor-bearing host may develop suppressor activities through the induc- tion of IL-10 gene expression (4, 19). In accord with these studies documenting the importance of lymphocyte-derived IL-10 in the generation of tumor-induced tolerance, we speculated that the unregulated overproduction of PGE2 by the tumor could be a central element in the immunosuppressive network because it leads to enhanced lymphocyte and macrophage IL-10 production.
IL-10 has the capacity to inhibit antitumor responses through several pathways. IL-10 has been documented to limit type 1 cytokine production (53, 54), Ag presentation (8, 55), and Ag-specific T cell proliferation (12). Pretreatment of tumor targets with IL-10 renders the tumor cells more resistant to CTL-mediated lysis (56, 57). IL-10 may also limit the access of functional APC to the tumor site (14). We have previously found that production of IL-10 by cutaneous carcinomas provides a mechanism for evasion of the local T cell immune response (13). We also found that transgenic mice overexpressing IL-10 under the control of the IL-2 promoter were unable to limit the growth of immunogenic tumors (49). Administration of blocking IL-10 mAbs restored in vivo antitumor responses in these transgenic mice. These findings support the suggestion that enhanced lymphocyte-derived IL-10 production antagonizes antitumor immunity (19). We have previously reported that NSCLC-derived PGE2 increases the transcriptional rate of IL-10 mRNA in PBL (4). We now report that COX-2 inhibition is an effective intervention to decrease the induction of IL-10 in the tumor-bearing host. The significance of the COX-2 inhibitor-induced decline in IL-10 production is demonstrated by transfer of IL-10 overproducing lymphocytes to tumor-bearing mice; transfer of these transgenic lymphocytes to normal mice overcomes the COX-2 inhibitor-induced antitumor response.
In addition to the modulation of IL-10 levels, we find that COX-2
inhibitor therapy results in up-regulation of IL-12. Macrophage IL-12
has previously been shown to be down-regulated in tumor-bearing mice
(18), and administration of this cytokine has been shown
to have antitumor effects (58, 59, 60, 61, 62). Elevated levels of
IL-12 may promote increased type 1 cytokine release and thus enhance
cell-mediated antitumor immune responses. In addition, through its
induction of IFN-
, IL-12 may serve to limit angiogenesis (63, 64).
However, other nonimmune-mediated mechanisms also may be operative. For example, overexpression of COX-2 can cause tumor cells to resist apoptosis (26, 65). In addition, COX-2 expression in colon tumor lines enhances tumor invasiveness (26), increases metastatic potential (27), and promotes angiogenesis (28). A recent study in gastric carcinomas found that COX-2 overexpression significantly correlated with tumor invasion into lymphatic vessels and metastasis to lymph nodes (66). In our current studies, although the proliferation of 1ASE7-3LL in vitro was comparable to that of the parental cell line, these COX2 antisense transfectants showed a marked decrease in tumorigenicity in vivo. Furthermore, mice receiving IL-10 transgenic lymphocytes evidenced a reversal of the antitumor effects of COX-2 inhibition. Thus, in addition to promoting a malignant phenotype, our findings suggest that COX-2 also plays an important role in antagonizing host immune reactivity against malignant cells.
Despite therapeutic efforts, lung cancer remains the major cause of cancer-related death in the United States (67). Although immunologic-based therapies have shown some success for other malignancies, lung cancer has been largely unresponsive (68). The lung tumor environment promotes immunosuppressive networks and the development of tolerance (5, 69). Our current findings identify tumor COX-2 expression as a critical element in the development of immunosuppression and documents that in vivo abrogation of this enzyme causes significant tumor reduction. Tumor-derived PGs play an important role in augmenting production of inhibitory cytokines such as IL-10 (4, 21) while suppressing endogenous production of cytokines including IL-12 that are necessary for effective host cell-mediated antitumor immune responses (20, 70). New therapies are needed for NSCLC (68), and our current findings suggest potential new avenues for therapeutic intervention. COX-2 is a focal point of immune-mediated PG production and thus a potential early point of intervention in attempts to restore effective cell-mediated immune responses in the tumor microenvironment. Our current findings add to our understanding of the complex interaction between pulmonary tumor-derived PG and the cytokine network at the tumor site. Tumor COX-2 expression may be an important therapeutic target for pharmacological or gene therapy intervention in NSCLC.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steven M. Dubinett, Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of California, Los Angeles, School of Medicine and West Los Angeles Veterans Affairs Medical Center, W111Q, 11301 Wilshire Boulevard, Los Angeles, CA 90073. E-mail address:
3 Abbreviations used in this paper: COX, cyclooxygenase; SSC, sodium chloride sodium citrate; EIA, enzyme immunoassay; NSCLC, non-small cell lung cancer; Lewis lung carcinoma cell line; CM, control medium.
Received for publication July 6, 1999. Accepted for publication October 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. T. Mao, I-H. Tsu, S. M. Dubinett, B. Adams, T. Sarafian, F. Baratelli, M. D. Roth, and K. J. Serio Modulation of Pulmonary Leukotriene B4 Production by Cyclooxygenase-2 Inhibitors and Lipopolysaccharide Clin. Cancer Res., October 15, 2004; 10(20): 6872 - 6878. [Abstract] [Full Text] [PDF]
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M. Pold, K. Krysan, A. Pold, M. Dohadwala, N. Heuze-Vourc'h, J. T. Mao, K. L. Riedl, S. Sharma, and S. M. Dubinett Cyclooxygenase-2 Modulates the Insulin-Like Growth Factor Axis in Non-Small-Cell Lung Cancer Cancer Res., September 15, 2004; 64(18): 6549 - 6555. [Abstract] [Full Text] [PDF]
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M. Mitsuhashi, J. Liu, S. Cao, X. Shi, and X. Ma Regulation of interleukin-12 gene expression and its anti-tumor activities by prostaglandin E2 derived from mammary carcinomas J. Leukoc. Biol., August 1, 2004; 76(2): 322 - 332. [Abstract] [Full Text] [PDF]
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H. E. Zeytin, A. C. Patel, C. J. Rogers, D. Canter, S. D. Hursting, J. Schlom, and J. W. Greiner Combination of a Poxvirus-Based Vaccine with a Cyclooxygenase-2 Inhibitor (Celecoxib) Elicits Antitumor Immunity and Long-Term Survival in CEA.Tg/MIN Mice Cancer Res., May 15, 2004; 64(10): 3668 - 3678. [Abstract] [Full Text] [PDF]
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S.-C. Yang, S. Hillinger, K. Riedl, L. Zhang, L. Zhu, M. Huang, K. Atianzar, B. Y. Kuo, B. Gardner, R. K. Batra, et al. Intratumoral Administration of Dendritic Cells Overexpressing CCL21 Generates Systemic Antitumor Responses and Confers Tumor Immunity Clin. Cancer Res., April 15, 2004; 10(8): 2891 - 2901. [Abstract] [Full Text] [PDF]
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M. Pold, L. X. Zhu, S. Sharma, M. D. Burdick, Y. Lin, P. P. N. Lee, A. Pold, J. Luo, K. Krysan, M. Dohadwala, et al. Cyclooxygenase-2-Dependent Expression of Angiogenic CXC Chemokines ENA-78/CXC Ligand (CXCL) 5 and Interleukin-8/CXCL8 in Human Non-Small Cell Lung Cancer Cancer Res., March 1, 2004; 64(5): 1853 - 1860. [Abstract] [Full Text] [PDF]
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M. S. Shaik, A. Chatterjee, and M. Singh Effect of a Selective Cyclooxygenase-2 Inhibitor, Nimesulide, on the Growth of Lung Tumors and Their Expression of Cyclooxygenase-2 and Peroxisome Proliferator- Activated Receptor-{gamma} Clin. Cancer Res., February 15, 2004; 10(4): 1521 - 1529. [Abstract] [Full Text] [PDF]
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S. Hillinger, S.-C. Yang, L. Zhu, M. Huang, R. Duckett, K. Atianzar, R. K. Batra, R. M. Strieter, S. M. Dubinett, and S. Sharma EBV-Induced Molecule 1 Ligand Chemokine (ELC/CCL19) Promotes IFN-{gamma}-Dependent Antitumor Responses in a Lung Cancer Model J. Immunol., December 15, 2003; 171(12): 6457 - 6465. [Abstract] [Full Text] [PDF]
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J. T. Mao, M. D. Roth, K. J. Serio, F. Baratelli, L. Zhu, E. C. Holmes, R. M. Strieter, and S. M. Dubinett Celecoxib Modulates the Capacity for Prostaglandin E2 and Interleukin-10 Production in Alveolar Macrophages from Active Smokers Clin. Cancer Res., December 1, 2003; 9(16): 5835 - 5841. [Abstract] [Full Text] [PDF]
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P. DeLong, T. Tanaka, R. Kruklitis, A. C. Henry, V. Kapoor, L. R. Kaiser, D. H. Sterman, and S. M. Albelda Use of Cyclooxygenase-2 Inhibition to Enhance the Efficacy of Immunotherapy Cancer Res., November 15, 2003; 63(22): 7845 - 7852. [Abstract] [Full Text] [PDF]
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J. E. Castelao, R. D. Bart III, C. A. DiPerna, E. M. Sievers, and R. M. Bremner Lung cancer and cyclooxygenase-2 Ann. Thorac. Surg., October 1, 2003; 76(4): 1327 - 1335. [Abstract] [Full Text] [PDF]
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G. Ferrandina, F. O. Ranelletti, F. Legge, L. Lauriola, V. Salutari, M. Gessi, A. C. Testa, U. Werner, P. Navarra, G. Tringali, et al. Celecoxib Modulates the Expression of Cyclooxygenase-2, Ki67, Apoptosis-Related Marker, and Microvessel Density in Human Cervical Cancer: A Pilot Study Clin. Cancer Res., October 1, 2003; 9(12): 4324 - 4331. [Abstract] [Full Text] [PDF]
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E. G. Cohen, T. Almahmeed, B. Du, D. Golijanin, J. O. Boyle, R. A. Soslow, K. Subbaramaiah, and A. J. Dannenberg Microsomal Prostaglandin E Synthase-1 Is Overexpressed in Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., August 1, 2003; 9(9): 3425 - 3430. [Abstract] [Full Text] [PDF]
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N.K. Altorki, R.S. Keresztes, J.L. Port, D.M. Libby, R.J. Korst, D.B. Flieder, C.A. Ferrara, D.F. Yankelevitz, K. Subbaramaiah, M.W. Pasmantier, et al. Celecoxib, a Selective Cyclo-Oxygenase-2 Inhibitor, Enhances the Response to Preoperative Paclitaxel and Carboplatin in Early-Stage Non-Small-Cell Lung Cancer J. Clin. Oncol., July 15, 2003; 21(14): 2645 - 2650. [Abstract] [Full Text] [PDF]
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K. Saukkonen, C. Tomasetto, K. Narko, M.-C. Rio, and A. Ristimaki Cyclooxygenase-2 Expression and Effect of Celecoxib in Gastric Adenomas of Trefoil Factor 1-deficient Mice Cancer Res., June 15, 2003; 63(12): 3032 - 3036. [Abstract] [Full Text] [PDF]
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L. X. Zhu, S. Sharma, B. Gardner, B. Escuadro, K. Atianzar, D. P. Tashkin, and S. M. Dubinett IL-10 Mediates Sigma1 Receptor-Dependent Suppression of Antitumor Immunity J. Immunol., April 1, 2003; 170(7): 3585 - 3591. [Abstract] [Full Text] [PDF]
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S. Sharma, M. Stolina, S.-C. Yang, F. Baratelli, J. F. Lin, K. Atianzar, J. Luo, L. Zhu, Y. Lin, M. Huang, et al. Tumor Cyclooxygenase 2-dependent Suppression of Dendritic Cell Function Clin. Cancer Res., March 1, 2003; 9(3): 961 - 968. [Abstract] [Full Text] [PDF]
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N. Heuze-Vourc'h, L. Zhu, K. Krysan, R. K. Batra, S. Sharma, and S. M. Dubinett Abnormal Interleukin 10R{alpha} Expression Contributes to the Maintenance of Elevated Cyclooxygenase-2 in Non-Small Cell Lung Cancer Cells Cancer Res., February 15, 2003; 63(4): 766 - 770. [Abstract] [Full Text] [PDF]
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M. Yao, S. Kargman, E. C. Lam, C. R. Kelly, Y. Zheng, P. Luk, E. Kwong, J. F. Evans, and M. M. Wolfe Inhibition of Cyclooxygenase-2 by Rofecoxib Attenuates the Growth and Metastatic Potential of Colorectal Carcinoma in Mice Cancer Res., February 1, 2003; 63(3): 586 - 592. [Abstract] [Full Text] [PDF]
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R. K. Batra, Y. Lin, S. Sharma, M. Dohadwala, J. Luo, M. Pold, and S. M. Dubinett Non-Small Cell Lung Cancer-derived Soluble Mediators Enhance Apoptosis in Activated T Lymphocytes through an I{kappa}B Kinase-dependent Mechanism Cancer Res., February 1, 2003; 63(3): 642 - 646. [Abstract] [Full Text] [PDF]
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M. Dohadwala, R. K. Batra, J. Luo, Y. Lin, K. Krysan, M. Pold, S. Sharma, and S. M. Dubinett Autocrine/Paracrine Prostaglandin E2 Production by Non-small Cell Lung Cancer Cells Regulates Matrix Metalloproteinase-2 and CD44 in Cyclooxygenase-2-dependent Invasion J. Biol. Chem., December 20, 2002; 277(52): 50828 - 50833. [Abstract] [Full Text] [PDF]
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B. S. Zweifel, T. W. Davis, R. L. Ornberg, and J. L. Masferrer Direct Evidence for a Role of Cyclooxygenase 2-derived Prostaglandin E2 in Human Head and Neck Xenograft Tumors Cancer Res., November 15, 2002; 62(22): 6706 - 6711. [Abstract] [Full Text] [PDF]
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J. G. Edwards, S. P. Faux, S. M. Plummer, K. R. Abrams, R. A. Walker, D. A. Waller, and K. J. O'Byrne Cyclooxygenase-2 Expression Is a Novel Prognostic Factor in Malignant Mesothelioma Clin. Cancer Res., June 1, 2002; 8(6): 1857 - 1862. [Abstract] [Full Text] [PDF]
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M. Pold, M. Dohadwala, J. Luo, Y. Lin, and S. Dubinett Microarray Identifies Cyclo-oxygenase-2-Dependent Modulation of Insulin-like Growth Factor Binding Protein-3 in Non-small Cell Lung Cancer Cells Chest, March 1, 2002; 121(2009): 29S - 30S. [Full Text] [PDF]
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M. B. Hansen-Petrik, M. F. McEntee, B. Jull, H. Shi, M. B. Zemel, and J. Whelan Prostaglandin E2 Protects Intestinal Tumors from Nonsteroidal Anti-inflammatory Drug-induced Regression in ApcMin/+ Mice Cancer Res., January 1, 2002; 62(2): 403 - 408. [Abstract] [Full Text] [PDF]
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K. Yoshimatsu, D. Golijanin, P. B. Paty, R. A. Soslow, P.-J. Jakobsson, R. A. DeLellis, K. Subbaramaiah, and A. J. Dannenberg Inducible Microsomal Prostaglandin E Synthase Is Overexpressed in Colorectal Adenomas and Cancer Clin. Cancer Res., December 1, 2001; 7(12): 3971 - 3976. [Abstract] [Full Text] [PDF]
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J. Fujita, J. R. Mestre, J. B. Zeldis, K. Subbaramaiah, and A. J. Dannenberg Thalidomide and Its Analogues Inhibit Lipopolysaccharide-mediated Induction of Cyclooxygenase-2 Clin. Cancer Res., November 1, 2001; 7(11): 3349 - 3355. [Abstract] [Full Text] [PDF]
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S. Sharma, M. Stolina, L. Zhu, Y. Lin, R. Batra, M. Huang, R. Strieter, and S. M. Dubinett Secondary Lymphoid Organ Chemokine Reduces Pulmonary Tumor Burden in Spontaneous Murine Bronchoalveolar Cell Carcinoma Cancer Res., September 1, 2001; 61(17): 6406 - 6412. [Abstract] [Full Text] [PDF]
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K. Yoshimatsu, N. K. Altorki, D. Golijanin, F. Zhang, P.-J. Jakobsson, A. J. Dannenberg, and K. Subbaramaiah Inducible Prostaglandin E Synthase Is Overexpressed in Non-Small Cell Lung Cancer Clin. Cancer Res., September 1, 2001; 7(9): 2669 - 2674. [Abstract] [Full Text] [PDF]
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 H. W. Murray Clinical and Experimental Advances in Treatment of Visceral Leishmaniasis Antimicrob. Agents Chemother., August 1, 2001; 45(8): 2185 - 2197. [Full Text] [PDF]
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 P. Kalinski, P. L. Vieira, J. H. N. Schuitemaker, E. C. de Jong, and M. L. Kapsenberg Prostaglandin E2 is a selective inducer of interleukin-12 p40 (IL-12p40) production and an inhibitor of bioactive IL-12p70 heterodimer Blood, June 1, 2001; 97(11): 3466 - 3469. [Abstract] [Full Text] [PDF]
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K. Xu, T. Yen, and C. L. Geczy IL-10 Up-Regulates Macrophage Expression of the S100 Protein S100A8 J. Immunol., May 15, 2001; 166(10): 6358 - 6366. [Abstract] [Full Text] [PDF]
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M. Oshima, N. Murai, S. Kargman, M. Arguello, P. Luk, E. Kwong, M. M. Taketo, and J. F. Evans Chemoprevention of Intestinal Polyposis in the Apc{{Delta}}716 Mouse by Rofecoxib, a Specific Cyclooxygenase-2 Inhibitor Cancer Res., February 1, 2001; 61(4): 1733 - 1740. [Abstract] [Full Text]
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S. Kulkarni, J. S. Rader, F. Zhang, H. Liapis, A. T. Koki, J. L. Masferrer, K. Subbaramaiah, and A. J. Dannenberg Cyclooxygenase-2 Is Overexpressed in Human Cervical Cancer Clin. Cancer Res., February 1, 2001; 7(2): 429 - 434. [Abstract] [Full Text]
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 M. W. Knoferl, D. Jarrar, M. G. Schwacha, M. K. Angele, W. G. Cioffi, K. I. Bland, and I. H. Chaudry Severe hypoxemia in the absence of blood loss causes a gender dimorphic immune response Am J Physiol Cell Physiol, December 1, 2000; 279(6): C2004 - C2010. [Abstract] [Full Text] [PDF]
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L. X. Zhu, S. Sharma, M. Stolina, B. Gardner, M. D. Roth, D. P. Tashkin, and S. M. Dubinett {Delta}-9-Tetrahydrocannabinol Inhibits Antitumor Immunity by a CB2 Receptor-Mediated, Cytokine-Dependent Pathway J. Immunol., July 1, 2000; 165(1): 373 - 380. [Abstract] [Full Text] [PDF]
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 S. M. Dubinett, R. K. Batra, P. W. Miller, and S. Sharma Tumor Antigens in Thoracic Malignancy Am. J. Respir. Cell Mol. Biol., May 1, 2000; 22(5): 524 - 527. [Full Text]
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M. Dohadwala, J. Luo, L. Zhu, Y. Lin, G. J. Dougherty, S. Sharma, M. Huang, M. Pold, R. K. Batra, and S. M. Dubinett Non-small Cell Lung Cancer Cyclooxygenase-2-dependent Invasion Is Mediated by CD44 J. Biol. Chem., June 8, 2001; 276(24): 20809 - 20812. [Abstract] [Full Text] [PDF]
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