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Annals of Surgical Oncology 10:305-313 (2003)
© 2003 Society of Surgical Oncology

ORIGINAL ARTICLES

Cyclooxygenase-2 Inhibition Improves Macrophage Function in Melanoma and Increases the Antineoplastic Activity of Interferon {gamma}

Michael Duff, AFRCSI, Philip P. Stapleton, PhD, Juan R. Mestre, MD, Sirish Maddali, MD, Gordon P. Smyth, FRCSI, Zhaoping Yan, MD, Tracy A. Freeman, BA and John M. Daly, MD, FACS

From the Weill Medical College of Cornell University/New York Presbyterian Hospital, Department of Surgery, New York, New York.

Correspondence: Address correspondence and reprint requests to: Philip P. Stapleton, PhD, Departments of Surgery and Microbiology/Immunology, Temple University School of Medicine, 3400 North Broad St., Room 513 OMS, Philadelphia, PA 19140; Fax: 215-707-8820; E-mail: ppstap{at}temple.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Melanoma inhibits macrophage tumoricidal activity and increases the expression of cyclooxygenase-2 (COX-2). In this study, we sought to determine whether inhibition of COX-2 could restore macrophage function and hence maximize the antitumor activity of the immune stimulant interferon {gamma} (IFN{gamma}).

Methods: Peritoneal macrophages were exposed to B16 melanoma-conditioned medium for 24 hours with or without the COX-2 inhibitor NS-398 and then were stimulated with lipopolysaccharide and IFN{gamma}. Cytotoxic activity, nitrite production, and cytokine production by the stimulated macrophages were measured. In addition, B16 melanoma cells were implanted intradermally into mice treated with IFN{gamma} (14,000 U on alternate days) alone or with a combination of IFN{gamma} and a COX-2 inhibitor (NS-398 or nimesulide). Mice were assessed for tumor growth and survival.

Results: Macrophage cytotoxicity and nitrite production were significantly suppressed by melanoma-conditioned medium (P < .01). This was prevented by 200 µM of NS-398 (P < .05). In vivo, combined treatment with IFN{gamma} and a COX-2 inhibitor caused a significant inhibition of tumor growth (P < .01) and improved survival (P = .02) compared with controls.

Conclusions: COX-2 inhibition reversed melanoma-induced suppression of macrophage function, and combined treatment of IFN{gamma} plus a COX-2 inhibitor was maximally effective in reducing tumor growth and improving survival.

Key Words: Melanoma • COX-2 • Macrophage • Interferon {gamma} • Nitric oxide • NS-398


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The incidence of melanoma is increasing among fair-skinned populations worldwide at a rate of 3% to 7% annually.1 In the United States, melanoma is the seventh most common malignancy, with a cumulative lifetime risk estimated at 1:75.2 Although case-related mortality has decreased in recent years, this is at a lower rate than the increase in incidence, and therefore overall mortality from melanoma continues to increase. Although early-stage disease is curable by surgical resection, survival rates decrease dramatically with advancing stage. The predicted 5-year survival is 46% with a tumor >4 mm thick, 40% with nodal disease, and <10% with metastatic disease.3,4 The median survival for patients with distant metastases is only 6 to 9 months.3

The dismal prognosis associated with advanced disease is partly due to the resistance of melanoma to currently available antineoplastic regimens. In recent years, emphasis has shifted toward immunotherapy, either through nonspecific immune stimulants such as interferon alfa (IFN{alpha}) and interleukin (IL)-2 or with vaccine therapy. Unfortunately, the results to date have been disappointing, with overall tumor response rates of <20%, and only one trial (Eastern Cooperative Oncology Group 1684) showed an improvement in overall survival.4,5

One explanation for the poor response of melanoma to immune therapy is the potential cellular immune-modulating effects of the tumor itself. The correlation between advancing malignant disease and diminished host immune response is well established and is seen clinically by the susceptibility of cancer patients to infectious complications.6,7 We have focused our work on macrophage function as a component of the host cellular defense system to melanoma growth.

Previous published work from this laboratory demonstrated a reduction in the cytotoxic activity of peritoneal macrophages elicited from subcutaneous (SC) tumor–bearing mice.8 These results correlated with reduced production of nitric oxide, tumor necrosis factor (TNF){alpha}, and superoxide anion. In addition, in vitro experiments examining the effect of exposure of macrophages to melanoma-conditioned media showed that changes in macrophage phenotype were associated with increased cyclooxygenase-2 (COX-2) activity and production of prostaglandin E2 (PGE2).9

The purpose of these studies was to examine the effect of melanoma-conditioned media on the cytotoxic activity of stimulated macrophages in the presence and absence of a COX-2 inhibitor. An in vivo model evaluated tumor growth in mice to elucidate whether the inhibition of PGE2 production could potentiate the antitumor effects of the immune stimulant IFN{gamma}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor
The B16 melanoma cell line (Tumor Depository, Division of Cancer Treatment, National Cancer Institute, Frederick, MD) is a nonmetastasizing, nonimmunogenic malignant melanoma that arises spontaneously in C57BL/6 mice. Cells were maintained in culture in Dulbecco’s modified Eagle medium (DMEM; Invitrogen Corp., Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and 1% antimicrobial solution (100 U/mL of penicillin, 100 µg/mL of streptomycin, .25 µg/mL of amphotericin B, and 50 µM of 2-mercaptoethanol) (Invitrogen), at 37°C and 5% CO2. The cell line was tested by the American Type Culture Collection (Manassas, VA) and Microbiological Associates (Rockville, MD) and was determined to be free of Mycoplasma organisms and common viruses and microorganisms.

Melanoma-Conditioned Media
Cultured B16 cells were dislodged by exposure to .05% trypsin solution, washed in DMEM, resuspended in DMEM/F12 at a concentration of 1 x 106 cells per milliliter, and maintained at 37°C. The supernatant was harvested after 24 hours, and suspended cells were removed by centrifugation. Before use, the conditioned medium was diluted to 20% in DMEM containing 3% FBS.

Animals
All experiments were performed with virus-free female C57BL/6 mice aged 6 to 8 weeks. Mice were allowed food and water ad libitum and left to acclimate for 1 week before use. Mice were housed in a facility approved by the Association for the Assessment and Accreditation of Laboratory Care Animals, and the Institutional Animal Care and Use Committee at Weill Medical College of Cornell University approved all animal protocols.

Peritoneal Elicited Macrophages
Peritoneal macrophages were elicited after intraperitoneal (IP) injection of thioglycollate. A 4% thioglycollate solution was prepared and sterilized by autoclave. Mice (n = 20 per experiment) were injected IP with 2 mL of thioglycollate solution. After 4 days, the mice were killed by CO2 asphyxiation, and macrophages were harvested by peritoneal lavage with calcium- and magnesium-free Hanks’ balanced salt solution (HBSS; Invitrogen). Elicited cells were washed and resuspended in complete HBSS. Viability was assessed by trypan blue exclusion, and cells were plated at 2 x 106 cells per milliliter in 6-well (2 mL per well) or 96-well (100 µl per well) dishes. After a 1-hour incubation at 37°C, plates were washed thoroughly with HBSS to remove all nonadherent cells. Adherent cells were >90% macrophages, as determined by functional and morphological criteria.10

Macrophage Functional Assays
For in vitro studies, isolated macrophages were divided into six groups (labeled C, C10, C200, B, B10, and B200). The groups labeled C were exposed to control media (DMEM with 3% FBS and 20% DMEM/F12) for 24 hours before stimulation. The groups labeled B were exposed to 20% B16 melanoma-conditioned media for 24 hours before stimulation. In groups C10 and B10, the media contained 10 µM of NS-398 (Cayman Chemical Co., Ann Arbor, MI). In groups C200 and B200, the media contained 200 µM of NS-398. After 24 hours of incubation, the media were removed; the macrophages were washed once with HBSS and then stimulated by the addition of DMEM containing 100 ng/mL of lipopolysaccharide (LPS) from Escherichia coli (strain O55:B5 from Sigma, St. Louis, MO) and 100 U/mL of IFN{gamma} (R&D Systems, Minneapolis, MN). Previous dose-response studies had demonstrated maximal nitrite production from macrophages stimulated at these doses. Experiments were repeated with an alternative COX-2 inhibitor, nimesulide (Cayman Chemical Co.).

Nitric Oxide and Cytokines
After 24 hours of stimulation, the supernatants were removed and assayed for nitrite levels by the Greiss reaction.11 Briefly, in a 96-well plate, 100 µl of medium was reacted with 100 µl of an equal mixture of 1% sulfanilamide and .1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid and incubated at room temperature for 10 minutes. Absorbance at 550 nm was measured by using a UVmax microplate reader (Molecular Devices, Menlo Park, CA), and data were calculated by extrapolating to standard curves generated with sodium nitrite standards.

Levels of TNF{alpha}, IL-6, IL-10, IL-12 p40, and IL-12 p70 were measured in the same supernatant samples by enzyme-linked immunosorbent assay (R&D Systems). Plates were read in a microplate reader, and concentrations were calculated from a generated standard curve by using DeltaSoft analysis software (Biometallics Inc., Princeton, NJ). Cytokine concentrations were expressed per microgram of protein in cell lysate.

Protein Determination
The BCA protein assay (Pierce, Rockford, IL) was used to quantify protein content per well. Briefly, after removal of the supernatant, 100 µl of distilled H2O was added to each well, and the plates were stored at -80°C. Protein content was determined after one freeze-thaw cycle, and values were estimated by extrapolation onto a standard curve by using bovine serum albumin as the standard protein. To control for variations in cell number, equivalent numbers of cells were used for each experiment, and after the adherence step, all results were normalized to cellular protein content.

Cytotoxicity
Tumoricidal activity was analyzed by measuring lactate dehydrogenase (LDH) production after coculture of isolated macrophages with B16 melanoma cells. Adherent macrophages (2 x 105 in 96-well dishes) were stimulated with IFN{gamma} and LPS for 16 hours, and then the medium was replaced. B16 cells were added to the wells at a melanoma:macrophage ratio of 1:20. Cells were then incubated for 48 hours, and the supernatant was harvested. Macrophage-mediated cytotoxicity was assayed by measuring the amount of LDH released from the lysed target cells by the method of Decker and Lohmann-Mattes.12 Specific lysis was calculated according to the formula equation


(1)

where A is the absorbance at 490 nm subtracted from the background absorbance (medium alone). A(experimental) is the net absorbance from wells containing activated macrophages and melanoma cells. A(spontaneous) represents the net absorbance from cells containing melanoma cells alone, and A(maximal) represents the net absorbance of cells that have been freeze-thawed to lyse all the cells; it represents the total amount of LDH that can be released from melanoma targets.

In Vivo Experiments
Tumor Growth
B16 melanoma cells were suspended in HBSS at 2 x 106 cells per milliliter. C57BL/6 mice (n = 33) each received 150 µl of the cell suspension as an intradermal injection in the interscapular region. Twenty-four hours after tumor implantation, mice were randomized into four groups: control (n = 10), NS-398 only (n = 6), IFN{gamma} only (n = 8), and NS-398 plus IFN{gamma} (n = 10). NS-398 was administered as an IP injection twice daily at a dose of 10 mg/kg. IFN{gamma} was administered at 14,000 U three times a week (Monday, Wednesday, and Friday), as an SC injection, at a site remote from the tumor. Control animals received vehicle (dimethyl sulfoxide [DMSO] in HBSS IP and HBSS SC) at the same time intervals. This dosing schedule for NS-398 has been demonstrated in previous experiments performed in this laboratory to provide adequate blockade of COX-2 activity.13 The dosing schedule for IFN{gamma} was chosen after preliminary experiments demonstrated maximal antitumor activity without significant toxicity at this dose.

Mice were maintained on regular diet and examined daily for evidence of tumor growth. Once palpable, tumors were measured every second day by using Verniers calipers. The examiner measuring the tumors was blinded to the treatment groups. Tumor diameter was measured in three perpendicular planes. Tumor volume was calculated by using the formula for the volume of an ellipsoid: equation


(2)

where a, b, and c represent the diameters in millimeters.

The experiment was terminated when some of the tumors became ulcerated, and on day 16, all mice were killed by CO2 asphyxiation. Tumors were dissected free and weighed. Tumor samples from two tumors in each group were snap-frozen and homogenized in a small volume of .9% saline. These samples were analyzed for PGE2 content by enzyme immunoassay according to the manufacturer’s protocol (Cayman Chemical Co.).

Survival
A second study set out to determine the effect of COX-2 inhibition on the survival of melanoma-bearing animals treated with IFN{gamma}. For this study, the selective COX-2 inhibitor nimesulide was given. This compound has similar properties to NS-39814 and can either be given by the IP route or prepared in the diet. The purpose of changing the drug and dosing schedule for this part of the study was to allow for higher doses of the COX-2 inhibitor to be administered while avoiding the trauma to the animals of twice-daily IP injections. To allow for maximal COX-2 inhibition at the time of stimulation with IFN{gamma}, mice in the combined-treatment group received a constant dose of nimesulide in the diet and then a bolus dose IP before each injection of IFN{gamma}.

The diet C11260 was prepared by Research Diets Inc. (Brunswick, NJ) and consisted of nimesulide at 400 ppm15 in Purina 5001 (Purina LabDiet, St. Louis, MO). Nimesulide was also prepared for IP injection by preparing a .1 M stock solution in DMSO, which was stored at -20°C and dissolved in HBSS for use. Mice randomized to receive the COX-2 inhibitor were commenced on the C11260 diet 1 day after injection of the tumor.

On Monday, Wednesday, and Friday, these animals also received an IP injection of nimesulide at 20 mg/kg of body weight. This was given 6 hours before the administration of IFN{gamma}. Control animals were fed the Purina 5001 diet without nimesulide and given an IP injection of an equivalent dose of DMSO on Monday, Wednesday, and Friday. IFN{gamma} was prepared and administered as described in the previous section. All animals were weighed at the start of the experiment, and their food intake was calculated daily.

B16 melanoma cells were suspended in HBSS at 2 x 106 cells per milliliter. C57BL/6 mice (n = 40) each received 150 µl of the cell suspension (3 x 106 cells) as an intradermal injection in the interscapular region. Twenty-four hours after tumor implantation, mice were randomized into the four treatment groups (n = 10 per group): the control group received a normal diet, an IP injection of DMSO on Monday, Wednesday, and Friday, and an SC injection of saline 6 hours after the DMSO; the IFN{gamma}-only group received the normal diet and DMSO but received the SC injection of IFN{gamma} (14,000 U) on Monday, Wednesday, and Friday; the nimesulide-only group received the C11260 diet and IP injections of nimesulide and SC saline on Monday, Wednesday, and Friday; and the combined-treatment group received the C11260 diet, IP nimesulide on Monday, Wednesday, and Friday, and IFN{gamma} as an SC injection 6 hours later.

The end point of this study was death of the animal, tumor ulceration, or excessive tumor burden as determined by both tumor size and the condition and behavior of the animal. The decision to kill an animal was taken by an independent observer, blinded to the treatment groups.

Histology
At death of the mouse, representative samples were taken from three tumors from each treatment group. The specimens were fixed in formalin, paraffin-embedded, and sectioned at 10 µm. Slides were stained with hematoxylin and eosin and examined under a light microscope at x10 and x40 magnification.

Statistics
Results of in vitro experiments and data regarding tumor growth were analyzed by analysis of variance. A P value of <.05 was regarded as significant. Results are presented as mean ± SD. Kaplan-Meier survival curves were generated and analyzed by the Breslow-Gehan-Wilcoxon test by using the StatView 4.11 statistics program (Abacus Concepts Inc., Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Melanoma-Induced Reduction in Macrophage Cytotoxicity Is Reversed by NS-398
To determine the effect of melanoma-conditioned media on macrophage-mediated killing of tumor cells, peritoneal elicited macrophages were activated by LPS 100 ng/mL and IFN{gamma} 100 U/mL, and cytotoxicity was measured. Figure 1A demonstrates that macrophages exposed to melanoma-conditioned media (group B) had significantly less mean cytotoxic activity against syngenic B16 melanoma targets when compared with macrophages cultured in control media (group C). The addition of 200 µmol of NS-398 to the media significantly reversed this effect (group B200).



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  		FIG. 1. Results of in vitro experiments (mean of three experiments ± SD) are shown. (A) There was a significant reduction in mean macrophage cytotoxic activity against B16 melanoma cells after prior incubation in melanoma-conditioned media (MCM) (C vs. B). The addition of NS-398 at 200 µM prevented this reduction (B vs. B200). (B) Incubation with MCM caused a significant reduction in macrophage mean nitric oxide (NO) production (C vs. B), and this effect was prevented by the addition of 200 µmol of NS-398 (B vs. B200). *P < .001; **P = .02. LDH, lactate dehydrogenase.

 
Melanoma-Induced Reduction in Macrophage Nitric Oxide Is Reversed by NS-398 (200 µmol)
After the demonstration of reduced cytotoxicity in macrophages exposed to melanoma-conditioned medium, we examined the effect of conditioned media on one of the major effector molecules of macrophage function, nitric oxide. By using the same six groups as for the cytotoxicity experiment, macrophage supernatant was collected 24 hours after stimulation and analyzed for nitrite content. The results, shown in Fig. 1B, demonstrate a significant reduction in mean nitric oxide production from stimulated macrophages after exposure to melanoma-conditioned medium (group B) compared with the control group (group C). This reduction was significantly reversed in the presence of the higher dose of NS-398 (group B200).

Melanoma Induces the T-Helper 2 Cytokine Profile in Macrophages
After 24 hours of stimulation with LPS and IFN{gamma}, the mean production of the cytotoxic molecule TNF{alpha} and the T-helper 1 cytokine IL-12 by the macrophages was significantly reduced by prior incubation with melanoma-conditioned media. In contrast, the mean production of T-helper 2 cytokines IL-10 and IL-6 was increased (Fig. 2). Although the changes in mean TNF{alpha} and IL-6 production were reversed by COX-2 inhibition, co-incubation with NS-398 did not alter the balance between IL-10 and IL-12.



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  		FIG. 2. These five charts demonstrate the effect of melanoma-conditioned media, with or without 200 µM of NS-398, on mean cytokine production by stimulated macrophages. When compared with controls (C), incubation with conditioned media (B) reduced macrophage mean production of the T-helper type 1 cytokines (tumor necrosis factor [TNF]{alpha} and interleukin [IL]-12) and increased mean production of the T-helper type 2 cytokines (IL-10 and IL-6). The addition of NS-398 (B200) reversed the observed changes in mean TNF{alpha} and IL-6 production but did not have a significant effect on the balance between IL-10 and IL-12 secretion. *P < .05 vs. C; **P < .05 vs. B.

 
Tumor Model Characteristics
Tumor Growth
Inoculation of B16 cells intradermally resulted in tumor growth in all mice. Animals were killed on day 16. Between day 14, when the final tumor volume measurement was made, and day 16, when tumors were harvested, four mice were killed because of ulcerated tumors. These included two mice from the control group, one from the IFN{gamma}-only group, and one from the combined-treatment group. These animals were therefore not included in the data on tumor weight. There was no difference in mean carcass weight between the groups at the time of death. Treatment with IFN{gamma}, NS-398, or vehicle control commenced 1 day after tumor inoculation and persisted until the end of the experiment.

Survival Study
Inoculation of B16 melanoma cells resulted in tumor growth in all animals. Mean animal weight and dietary intake were constant between the groups. Animals receiving the treatment diet consumed an average of 2.65 g of food per animal per day over the first 2 weeks of the experiment, compared with 2.55 g per animal per day in the control diet groups. The mean weight of the animals in the nimesulide-receiving groups was 20.2 g, compared with a mean weight of 20.0 g in the other two groups. The concentration of nimesulide in the treatment diet was 400 ppm, so the mean dose of nimesulide received by the mice given this diet was 52.7 mg/kg/day.

IFN{gamma} and NS-398 Act Synergistically To Reduce Tumor Growth
The result of treatment on tumor growth is shown in Fig. 3. Tumors were measured with calipers from day 7, when 32 of 33 mice had palpable tumors. Although mean tumor growth was initially equal in all groups, by day 14 a significant reduction in mean tumor size was noted in mice that received combined treatment of NS-398 plus IFN{gamma} (P = .006). This reduction was greater than that seen with either treatment alone.



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  		FIG. 3. This graph plots mean tumor volume in the four groups against time. By 2 weeks after tumor implantation, there was a significant difference in mean tumor size between the control animals and those receiving combined therapy of interferon (IFN){gamma} plus NS-398. *P < .01.

 
After death of the mice, tumors were harvested and weighed. Mean tumor weights are shown in Fig. 4. Animals in the combined-treatment group had significantly smaller tumors than those in the control or NS-398–only groups (P < .01).



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  		FIG. 4. Mean tumor weights ± SD are shown. Sixteen days after implantation, tumors were dissected free and weighed. There was a significant difference in mean tumor weight between the combined-treatment group and controls (P < .01) and between controls and the interferon (IFN){gamma}–only group (P < .05).

 
To confirm the efficacy of COX-2 inhibition with NS-398 at 10 mg/kg twice daily, tumor samples (n = 2 per group) were analyzed for PGE2 content. The results (Fig. 5) show complete inhibition in the NS-398–only group, upregulation with IFN{gamma}, and partial inhibition with IFN{gamma} plus NS-398. This would suggest that a higher dose of NS-398 might be required for complete COX-2 inhibition when combined with IFN{gamma} treatment. Histological examination of the tumor specimens revealed no differences among the groups in overall composition, lymphocytic infiltration, or microvessel density.



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  		FIG. 5. Two tumors from each group were lysed and analyzed for levels of prostaglandin E2 (PGE2). The average value for each group is shown. NS-398 caused inhibition of PGE2 production, although this was not complete in the combined-treatment group.

 
IFN{gamma} and Nimesulide Act Synergistically To Improve Survival
Mice (10 per group) were implanted intradermally with 3 x 105 B16 melanoma cells and followed up for survival. Kaplan-Meier survival curves were constructed and analyzed by the Breslow-Gehan-Wilcoxon test (Fig. 6). Treatment with either IFN{gamma} or nimesulide alone led to a small, although not statistically significant, improvement in survival in these animals relative to the control group. However, treatment of the tumor-bearing animals with a combination of nimesulide and IFN{gamma} significantly improved the overall survival rate (P = .02) and increased the mean survival time from 18.4 ± 4.9 days to 24.8 ± 6.4 days (P = .0007).



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  		FIG. 6. Tumor-bearing mice were divided into four treatment groups and followed up for survival. Kaplan-Meier survival curves were plotted as shown. There was a significant difference (*P = .02) between the control and combined-treatment groups when they were compared by using the Breslow-Gehan-Wilcoxon test. There was no statistically significant difference between any of the other groups. IFN, interferon.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The macrophage forms an intrinsic part of the body’s primary defense against malignancy. The antitumor action of macrophages involves direct cell lysis, phagocytosis, cytokine release, and antigen presentation.16,17 The presentation of tumor antigens with major histocompatibility complex (MHC) class II molecules enables the development of specific immunity through the priming of CD4+ T cells. Presentation of an antigen with MHC class I molecules allows a cytotoxic T-lymphocyte response through the activation of CD8+ T cells. In addition, the macrophage promotes natural killer cell and cytotoxic T-lymphocyte activity through the release of T-helper type 1 cytokines, in particular, IL-12.

One of the key effector molecules for macrophage tumoricidal activity is nitric oxide, which is synthesized in macrophages by the inducible form of nitric oxide synthase (iNOS). The mechanisms of nitric oxide cytotoxicity are complex, involving inhibition of DNA synthesis, mitochondrial inactivation, cell membrane lysis, cell-cycle arrest, DNA strand break formation, and the induction of apoptosis. In addition, nitric oxide can react with superoxide to form peroxynitrite, a powerful oxidizing agent capable of inducing cell injury and death.18 Inducible NOS expression and, hence, nitric oxide production, is induced in macrophages in response to a variety of stimuli, in particular, the T-helper 1 cytokines IFN{gamma} and TNF{alpha} and the bacterial wall component LPS. Inhibition of nitric oxide production increases the host susceptibility to viral, bacterial, fungal, protozoal, and helminthic infections. In addition, the antitumor activity of stimulated mouse macrophages is absent in iNOS knock-out mice.19 Aside from its cytotoxic effects, nitric oxide is involved in regulating the activity of MHC class II expression in antigen-presenting cells, in modulating T-cell mitogenic responses, and in inducing and suppressing many cytokines.

For a tumor to grow and metastasize successfully, it must develop mechanisms of counteracting the host immune response. Indeed, through the production of immune-modulatory proteins, a tumor may actually manipulate the host immune cells to provide the growth factors, angiogenic factors, and proteases it requires for growth, invasion, and metastasis.20–22

In this study, we have examined in vivo and in vitro the effect of COX-2 inhibition on the interaction between melanoma and activated tumor-associated macrophages. We used IFN{gamma} to stimulate the macrophage response because it is a naturally occurring cytokine produced by natural killer cells and activated T-helper cells. The IFN{gamma} receptor is expressed on all nucleated cells, and the binding of IFN to its receptor activates an intracellular signaling cascade (the JAK-STAT pathway) leading to the binding of specific transcription factors (STAT-1{alpha} homodimer and IRF) and gene expression. IFN{gamma}, along with IL-2, IL-4, and IL-12, is classified as a T-helper type 1 cytokine. It promotes the bactericidal activity of phagocytes, induces iNOS transcription, and stimulates antigen presentation through MHC class I and II molecules.23

Despite promising reports of recombinant IFN{gamma} therapy in preclinical models, the results of systemic IFN{gamma} therapy in the treatment of patients with metastatic melanoma have been poor, with response rates as low as 5%.24,25 One potential explanation for the poor clinical response, despite demonstrable improvements in immune function, is the immunosuppressive effect of factors in the tumor environment. Inhibitory cytokines, including IL-10 and transforming growth factor ß, are produced by melanoma cells17 and are capable of inhibiting the cytotoxic activity of the host immune system. However, experiments using blocking antibodies to these proteins and other known inhibitory cytokines (MIC-1 and IL-4) failed to reverse the immune-suppressive effects of the B16 melanoma-conditioned media in our model.26

In this study, we have demonstrated that the selective COX-2 inhibitor NS-398 can effectively reverse the inhibition of macrophage function caused by melanoma-conditioned media. This was most clearly demonstrated by the changes in nitric oxide production and macrophage-mediated cytotoxicity against melanoma cells in vitro. In an in vivo model of SC melanoma growth, we demonstrated that the addition of NS-398 to a therapeutic dose of IFN{gamma} can lead to a further reduction in tumor growth when compared with IFN{gamma} therapy alone. In addition, a similar COX-2 inhibitor, nimesulide, when added to IFN{gamma}, was able to improve the overall survival of melanoma-bearing animals.

Studies examining the effects of human strains of melanoma on peripheral blood mononuclear cell function have also demonstrated alterations in monocyte function with an increase in COX-2 expression and PGE2 production,9 indicating a similarity between the human and murine models. As the search for effective immune-therapy regimens for the treatment of metastatic melanoma continues, we speculate that the addition of a selective COX-2 inhibitor, as an adjunct to immune-stimulatory agents, may increase their potency as antitumor agents.


   Footnotes
 
Treatment of macrophages with a cyclooxygenase-2 (COX-2) inhibitor reverses the suppressive effect of melanoma-conditioned media on cytotoxicity and nitric oxide production. An antineoplastic regimen combining COX-2 inhibition with immune-modulatory therapy with interferon-{gamma} was more effective than individual therapy in reducing tumor growth and improving survival in a murine model of melanoma.

Received for publication April 29, 2002. Accepted for publication October 24, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Armstrong BK. Cutaneous melanoma. Cancer Surv 1994; 19: 219–39.
  2. Marks R. Epidemiology of melanoma. Clin Exp Dermatol 2000; 25: 459–63.[CrossRef][Medline]
  3. Becker JC, Kampgen G, Brocker E. Classical chemotherapy for metastatic melanoma. Clin Exp Dermatol 2000; 25: 503–8.[CrossRef][Medline]
  4. Whittaker S. Adjuvant therapy in melanoma. Clin Exp Dermatol 2000; 25: 497–502.[CrossRef][Medline]
  5. Cole B, Kirkwood J, Goldhirsch A, et al. Quality of life adjusted survival for interferon {alpha}-2b treatment of high risk resected cutaneous melanoma: an Eastern Cooperative Oncology Group study. J Clin Oncol 1996; 14: 2666–73.[Abstract/Free Full Text]
  6. Louria DB. Controversies in the management of infectious complications of neoplastic disease. Introduction and epidemiology. Am J Med 1983; 76: 414.[CrossRef]
  7. Rosenberg AS, Brown AE. Infection in the cancer patient. Dis Month 1993; 39: 505–69.
  8. Naama H, Mack V, Smyth G, et al. Macrophage effector mechanisms in melanoma in an experimental study. Arch Surg 2001; 136: 804–9.[Abstract/Free Full Text]
  9. Eisengart CA, Naama HA, Mackrell PJ, et al. Prostaglandins regulate melanoma-induced cytokine production in macrophages. Cell Immunol 2000; 204: 143–9.[CrossRef][Medline]
  10. Adams DO, Koren HS. Methods for Studying Mononuclear Phagocytes. New York: Academic Press, 1981.
  11. Green LC, Ruiz de Lazuriaga K, Wagner DA, et al. Nitrate biosynthesis in man. Proc Natl Acad Sci U S A 1981; 83: 7764–8.
  12. Decker T, Lohmann-Matthes ML. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor-necrosis factor activity. J Immunol Methods 1988; 115: 61–9.[CrossRef][Medline]
  13. Strong VE, Concannon EM, Naama HA, et al. Blocking prostaglandin E2 after trauma attenuates pro-inflammatory cytokines and improves survival. Shock 2000; 14: 374–9.[Medline]
  14. Barnett J, Chow J, Ives D, et al. Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system. Biochim Biophys Acta 1994; 1209: 130–9.[CrossRef][Medline]
  15. Fukutake M, Nakatsugi S, Isoi T, et al. Suppressive effects of nimesulide, a selective inhibitor of cyclooxygenase-2, on azoxymethane-induced colon carcinogenesis in mice. Carcinogenesis 1998; 19: 1939–42.[Abstract/Free Full Text]
  16. Keller R, Keist R, Wechsler A, et al. Mechanisms of macrophage-mediated tumor cell killing: a comparative analysis of the roles of reactive nitrogen intermediates and tumor necrosis factor. Int J Cancer 1990; 46: 682–6.[Medline]
  17. Pak CC, Fidler IJ. Molecular mechanisms for activated macrophage recognition of tumor cells. Semin Cancer Biol 1991; 2: 189–95.[Medline]
  18. Burney S, Tamir S, Gal A, et al. A mechanistic analysis of nitric oxide-induced cellular toxicity. Nitric Oxide 1997; 1: 130–44.[CrossRef][Medline]
  19. MacMicking J, Xie XQ, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol 1997; 15: 323–50.[CrossRef][Medline]
  20. Mantovani A. Tumor-associated macrophages in neoplastic progression: a paradigm for the in vivo function of chemokines. Lab Invest 1994; 71: 5–16.[Medline]
  21. Meier FS, Satyamoorthy K, Nesbit M, et al. Molecular events in melanoma development and progression. Front Biosci 1998; 3: 1005–10.
  22. Montavani A, Colotta F, Sozzani S, et al. The origin and function of tumor-associated macrophages. Immunol Today 1992; 13: 265–70.[CrossRef][Medline]
  23. Boehm U, Klamp T, Groot M, et al. Cellular responses to interferon-gamma. Annu Rev Immunol 1997; 15: 749–95.[CrossRef][Medline]
  24. Schiller JH, Kirkwood JM, Karp D, et al. Eastern cooperative group trial of interferon gamma in metastatic melanoma: an innovative study design. Clin Cancer Res 1996; 2: 29–36.[Abstract/Free Full Text]
  25. Kirkwood JM, Schiller JH, Oken MM, et al. Immunomodulatory function of interferon-gamma in patients with metastatic melanoma: results of a phase II-B trial in subjects with metastatic melanoma, ECOG study E 4987. Eastern Cooperative Oncology Group. J Immunother 1997; 20: 146–57.
  26. Naama H, McCarter M, Mack V, et al. Suppression of macrophage nitric oxide production by melanoma: mediation by a melanoma-derived product. Melanoma Res 2001; 11: 229–38.[CrossRef][Medline]



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