Download Leukotriene B4 Creates a Favorable Microenvironment for Murine

Published OnlineFirst September 8, 2009; DOI: 10.1158/1541-7786.MCR-09-0038
Published Online First on September 8, 2009
Leukotriene B4 Creates a Favorable Microenvironment
for Murine Melanoma Growth
André Luis Lacerda Bachi,1 Fabiana Jin Kyung Kim,1 Suely Nonogaki,2
Célia Regina Whitaker Carneiro,1 José Daniel Lopes,1 Miriam Galvonas Jasiulionis,1,3
and Mariangela Correa1,4
1
Disciplina de Imunologia, Universidade Federal de São Paulo; 2Hospital do Câncer, Fundação Antônio
Prudente; 3Departamento de Farmacologia, Universidade Federal de São Paulo; and
4
Instituto do Câncer do Estado de São Paulo, São Paulo, SP, Brazil
Abstract
Chronic inflammation has long been associated with
neoplastic progression. Our group had recently shown that
the addition of a large number of apoptotic tumor cells to
the tumor microenvironment induces a potent acute
inflammatory reaction capable of promoting melanoma
growth; however, primarily necrotizing cells do not cause
such a reaction. Here, we show that potent inflammatory
agents, such as lipopolysaccharide (LPS) and carrageenan,
also promote growth of subtumorigenic doses of
melanoma cells, having no effect on melanoma proliferation
in vitro. Inhibition of 5-lipoxygenase (5-LOX) seems to have
a pivotal role in this model because caffeic acid and
MK886, a FLAP (5-LOX–activating protein) inhibitor, partially
hindered tumor growth induced by apoptotic cells or LPS.
Other enzymes of the arachidonic acid pathway,
cyclooxygenase-1 and cyclooxygenase-2, seem to have no
participation in this tumor promoter effect, as the inhibitor
of both enzymes (indomethacin) did not alter melanoma
growth. Leukotriene B4 (LTB4), the main product of
the 5-LOX pathway, was able to induce growth of
subtumorigenic inocula of melanoma cells, and a LTB4
receptor antagonist inhibited acute inflammation-associated
tumor growth. Addition to the tumor inflammatory
microenvironment of eicosapentaenoic acid, an
ω3-polyunsaturated fatty acid with anti-inflammatory
properties, or leukotriene B5, an eicosapentaenoic
acid–derived leukotriene, significantly inhibited tumor
development. These results give new insights to the
mechanisms through which inflammation may contribute to
tumor progression and suggest that LOX has an important
role in tumor progression associated with an inflammatory
state in the presence of apoptosis, which may be a
Received 1/28/09; revised 6/4/09; accepted 6/24/09; published OnlineFirst 9/8/09.
Grant support: Fundação de Amparo à Pesquisa do Estado de São PauloFAPESP, grant 06/61293-1 (M.G. Jasiulionis), and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior-CAPES, grant 33009015003P3
(M.G. Jasiulionis).
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Miriam Galvonas Jasiulionis, Universidade Federal de São
Paulo, Rua Botucatu, 862, 4° andar, São Paulo, SP 04023-900, Brazil. Phone:
55-11-5576-4529; Fax: 55-11-5572-3328. E-mail: [email protected]
Copyright © 2009 American Association for Cancer Research.
doi:10.1158/1541-7786.MCR-09-0038
Mol Cancer Res 2009;7(9). September 2009
consideration for apoptosis-inducing treatments, such as
chemotherapy and radiotherapy. (Mol Cancer Res 2009;
7(9):1417–24)
Introduction
The association between inflammation and cancer was noted
by Rudolph Virchow in 1858, when he described for the first
time the presence of a leukocytic infiltrate in tumor tissues and
concluded that cancers frequently occurred at sites of chronic
inflammation (1). The contribution of environmental factors
to cancer progression is becoming the focus of intense research,
and special attention has been devoted to chronic inflammatory
diseases that are associated with a high prevalence of tumors
(2, 3). Those include common conditions such as gastritis, esophagitis, prostatitis, and chronic skin ulcers, among others. Although several of these pathologies are associated with viral or
bacterial infections, many others are not, and the mechanisms
through which cancer develops in the inflammatory microenvironment are only beginning to be uncovered.
Opposing effects of inflammation on cancer have been described. Acute inflammation is believed to induce an antitumor
immune response, whereas chronic inflammation seems to facilitate neoplastic growth (4). Very few clinical reports corroborate
the first assumption, which seems to have been mostly based on
animal studies of tumor immunity, whereas several epidemiologic, clinical, and experimental studies in humans as well as experimental studies in animals confirm the second postulate (5).
Eicosanoids, 20-carbon lipid-derived effectors of the inflammatory response, play a central role in carcinogenesis promotion and environmental support of neoplastic progression
(6). These molecules, comprising several different classes
and functions, are mostly derived from membrane-associated
lipids, particularly arachidonic acid (AA), which is an ω6
polyunsaturated fatty acid (PUFA). There are two major metabolic pathways of interest in the AA cascade: the cyclooxygenase (COX) and the lipoxygenase (LOX) pathways. Both
pathways have direct effects on carcinogenesis induction and
apoptosis suppression (7, 8). Furthermore, both inflammatory
pathways are linked indirectly to the induction and promotion
of tumor growth through their actions on the microenvironment, such as induction of angiogenesis and growth factor
production (9).
Apoptosis, or programmed cell death, occurs at high rates in
infected, inflamed, or remodeling tissues. Free apoptotic cells
are rarely found in tissues, even during inflammation, as they
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are rapidly removed by either local nonprofessional phagocytes
or specialized phagocytes such as macrophages (10). Recognition, uptake, and degradation of apoptotic cells by phagocytes
occur extremely fast, preventing inappropriate exposure of adjacent tissues to the intracellular contents of the cells undergoing
apoptosis. A hallmark of the removal of apoptotic cells in vivo is
the failure to generate an associated inflammatory response.
Thus, depending on the context, the removal of apoptotic cells
may induce an inhibitory effect that can antagonize a coincident
proinflammatory response. In contrast, the uptake of necrotic
cells results in activation of the inflammatory response and
may promote an immune response to self-antigens (11, 12).
Based on the evidence found in our laboratory (13) that
showed the positive correlation between acute inflammation,
induced by massive apoptosis, and melanoma progression,
we extended our investigation on the inflammatory pathways
involved in that model.
Results
Different Acute Inflammatory Stimuli Promote Melanoma
Tumor Progression
An essential aspect in the homeostasis maintenance of a
multicellular organism is the disposal of dead cells, especially
apoptotic cells. Normally, this clearance process is not characterized by the induction of an inflammatory response. However,
several studies have shown that the death of a great amount of
cells by apoptosis in certain cases can lead to a secondary necrosis followed by the activation of an inflammatory response
(14, 15). We have previously observed (13) that the coinjection
of a large amount of apoptotic cells (106 γ-irradiated Tm1 melanoma cells) is associated with a local inflammatory response
and promotes the growth of a subtumorigenic dose of viable
melanoma cells (103 Tm1 cells; Fig. 1A). To confirm the key
role of the acute inflammatory process in the growth of this
subtumorigenic amount of melanoma cells, 103 Tm1 cells were
coinjected with known inflammatory stimuli, lipopolysaccharide (LPS) or carrageenan. Both LPS (Fig. 1B) and carrageenan
(Fig. 1C) are capable of promoting the growth of this suboptimal number of tumor cells, similarly to the coinjection protocol
using apoptotic and viable tumor cells (Fig. 1A). These data
hint at the presence of common inflammatory pathways involved in melanoma progression induced by these inflammatory stimuli. We chose to focus on the role of apoptotic cells as an
inflammatory stimulus because of potential clinical implications related to cancer treatments because radiotherapy and chemotherapy treatments induce massive apoptosis.
Apoptosis Occurs for at Least 72 Hours at the Injection
Site and Is Accompanied by an Inflammatory Infiltrate
Subcutaneous tissues coinjected with apoptotic (106) and viable tumor cells (103) were surgical removed 6 hours, 24 hours,
72 hours, and 14 days after coinjection. Histologic analysis
showed the presence of an intense cellular infiltrate surrounding the injection site after 6 and 24 hours, which was reduced
after 72 hours and almost absent 14 days after the coinjection
(Fig. 2, HE, top). A large number of dying cells, in varying
stages of the process, and including necrotic debris, were also
clearly noted. Terminal deoxynucleotidyl transferase–mediated
dUTP nick end labeling (TUNEL) analysis showed the pres-
FIGURE 1. Different acute inflammatory stimuli promote melanoma tumor progression. C57Bl/6 mice s.c. injected with a subtumorigenic dose of
103 Tm1 viable melanoma cells are able to form fast-growing tumors when
coinjected with 106 Tm1 apoptotic cells (A) or 2.5 μg LPS (B) or 0.1%
carrageenan (C). As a control group, animals were injected with 103
Tm1 melanoma viable cells (subtumorigenic dose). All experiments were
done using groups of five to ten 6- to 8-wk-old female mice, which were
considered harboring tumors when the subcutaneous mass reached 20
mm3 (palpable tumor). *, P < 0.05, statistical significance using nonpaired
nonparametric Student's t tests.
ence of apoptotic cells diffusely located in the injection site 6
and 24 hours after coinjection. After 72 hours, the apoptotic
cells are concentrated in the central region of the injection site,
surrounded by viable melanoma cells. After 14 days, we observed few apoptotic areas in the tumor mass (Fig. 2, TUNEL,
bottom) and very few inflammatory cells surrounding or permeating the tumor mass (Fig. 2, HE, top).
Inhibition of the 5-LOX Pathway, but not COX, Hinders
Tumor Progression Induced by Acute Inflammation
The recruitment of leukocytes and production of inflammatory mediators are important elements in any inflammatory
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LTB4 Favors Melanoma Progression
response. The metabolic pathway of activation and production
of these mediators includes the utilization of AA (an ω6 fatty
acid derived from linoleic acid obtained from the diet) or
eicosapentaenoic acid (EPA, an ω-3 fatty acid derived from
α-linolenic acid obtained from the diet), and enzymes such
as LOX or COX that convert these fatty acids in leukotrienes
or prostaglandins, respectively.
Animal groups submitted either to coinjection (106 apoptotic cells and 103 viable tumor cells) or injection of LPS
and 103 viable tumor cells or injection of a tumorigenic dose
of viable tumor cells only (in the concentration of 2 × 105
melanoma cells) were treated with indomethacin, a nonselective inhibitor of COX-1 and COX-2, caffeic acid (5-LOX inhibitor), or MK886 [FLAP (5-LOX–activating protein)
inhibitor]. Figure 3 shows that the use of indomethacin does
not interfere with tumor progression, as observed with the
COX-2–selective inhibitor meloxican (data not shown), suggesting that the activation of the cyclooxygenase pathway
does not play an important role in growth of a subtumorigenic dose of melanoma cells induced by a large number
of apoptotic cells (Fig. 3A) or by the potent inflammatory
agent LPS (Fig. 3G). The anti-inflammatory activity of these
inhibitors was previously confirmed by footpad inflammation
assays (data not shown). However, 5-LOX pathway inhibitors, such as caffeic acid (Fig. 3B and H) or MK886
(Fig. 3C), inhibit tumor progression in this model. Dexamethasone, a glucocorticosteroid with very complex effects
on inflammatory pathways (16, 17), also does not have
any effect on melanoma growth in vivo or in vitro (data
not shown). Tumor growth unrelated to acute inflammation
is not influenced by any of these inhibitors (Fig. 3D-F).
Neutrophil recruitment to inflamed sites is stimulated by
leukotriene B4 (LTB4), one of the main products of the
AA/5-LOX inflammatory pathway. Inhibition of this pathway
by caffeic acid, but not by COX inhibitors (data not shown),
altered neutrophil recruitment kinetic to the injection site
(Fig. 4A and B), confirming that, in this model, tumor
growth depends on the inflammatory response elicited by
the 5-LOX pathway but not on COX-mediated inflammation.
LTB4, a Product of the 5-LOX Pathway, Is Essential for
Melanoma Progression Associated with Acute
Inflammation
Leukotrienes, synthesized from AA or EPA through the 5LOX pathway, play a major role in inflammatory reactions.
Among the biologically active metabolites of this pathway,
LTB4, derived from leukotriene A4 (LTA4) in the AA/5-LOX
metabolic pathway, is a potent chemoattractant involved in the
recruitment of neutrophils and leukocytes to the inflamed sites
(18). To evaluate the role of LTB4 in this model, animals received a subtumorigenic dose of Tm1 cells (103 melanoma
cells) admixed with 100 nmol/L of LTB4 in the subcutaneous
tissue (Fig. 5A), whereas no effects of LTB4 on in vitro tumor
cell proliferation were observed (Fig. 5B). Conversely, mice
subjected to coinjection were treated with LTB4-ethanolamide
(Eth), a LTB4 receptor 1 (BLTR1) antagonist (19). Treatment
with LTB4-ethanolamide (Eth) significantly reduced tumor progression associated with inflammation (Fig. 5C), but did not
present any effect on tumor growth in animals injected only
with tumor cells (Fig. 5D).
EPA, an ω3 PUFA, and Its Derived Mediator Leukotriene
B5 Inhibit Melanoma Growth Associated with
Inflammation
EPA is an ω3 PUFA obtained from the diet and, similar to
AA, is a substrate for COX and LOX enzymes, among others.
Mediators derived from EPA present mostly anti-inflammatory
properties (20) and have beneficial effects on patients with cancer (reviewed in ref. 21). To study their role in our model,
groups of animals received s.c. EPA or leukotriene B5
(LTB5) close to the coinjection site. As shown in Fig. 6A
and C, both significantly reduced melanoma growth induced
by inflammation while presenting no effects on control groups
(Fig. 6B and D).
Discussion
Chronic infection and inflammation have long been associated with cancer, but only in recent years has this complex
correlation begun to be unraveled (22). Several experiments
FIGURE 2. Apoptosis occurs up to 72 h in parallel with acute inflammatory infiltrate. Histologic analysis (HE; top) of the coinjection site of 106 Tm1
apoptotic cells and 103 viable Tm1 melanoma cells 6 h, 24 h, 72 h, and 14 d after injection. Immunohistochemistry assay for apoptosis (TUNEL; bottom)
shows the presence of apoptotic cells along tumor progression (×100).
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FIGURE 3. Inhibition of the 5-LOX pathway, but not COX, reduces tumor progression associated with an inflammatory state. Induction of melanoma
growth (103 viable Tm1 cells) by apoptotic cells (106 γ-irradiated Tm1; A) or LPS (2.5 μg/animal; G) in the coinjection arm is not abrogated by indomethacin,
a COX-1/COX-2 inhibitor (20 mg/animal/d), but is reduced by treatment with caffeic acid, a 5-LOX inhibitor (25 mg/animal/d; B and H, respectively), and
MK886, a FLAP inhibitor (1 mg/kg/d; C). Treatment of animals with indomethacin (D), caffeic acid (E), or MK886 (F) does not affect the tumor progression
obtained with injection of 2 × 105 Tm1 melanoma cells (tumorigenic dose).
using animal models have clearly established a role for cells
of the immune system in tumor progression (23) and such
phenomenon in humans may be associated with a poor prognosis in the presence of tumor-associated macrophages (24),
or a favorable prognosis when associated to neutrophil infiltration (reviewed in ref. 25), although several other reports do
not support the last conclusion (26-31). Although human melanoma is not generally associated with overt inflammation,
several recent studies highlight the importance of an inflammatory microenvironment, especially the role of the innate
immunity, for melanoma initiation and progression (reviewed
in ref. 32). Proteomic analysis of B16F10 mouse melanoma
suggested that inflammatory proteins are overexpressed in
the first 3 to 7 days of tumor challenge in C57Bl-6 mice
(33), and, perhaps more interestingly, an inflammatory gene
signature was able to identify with high accuracy human metastatic melanoma present in sentinel lymph nodes but not in
nonmetastatic nodes (34).
Our results show that the generation of an acute inflammatory process induced by apoptotic cells, LPS, or carrageenan
provides a favorable environment for the development of tumors when a subtumorigenic dose of melanoma cells is s.c.
injected in mice (Fig. 1). Although apoptosis has been associated with the inhibition of inflammatory responses, several
factors, including unscheduled and massive cell loss, contribute to the “decision” by macrophages of which type of response is adequate at the moment (35). Furthermore, it is
believed that the type of cell death (necrotic versus apoptotic)
is also determined by the type and amount of tissue injury,
and concurrent apoptosis of a large number of cells, with
an associated clearance deficiency, may lead to secondary necrosis and a proinflammatory response (36, 37). Effective cancer therapy (chemotherapy, radiotherapy, immunotherapy)
induces activation of apoptosis programs (38), but little is
known about the host response to apoptotic cells in the tumor
microenvironment. Figure 2 and a previous work by our
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LTB4 Favors Melanoma Progression
group (13) show that irradiated melanoma cells undergo massive apoptosis when injected s.c., which is accompanied by a
leukocytic infiltrate indicative of an inflammatory response.
The inflammatory infiltrate peaks 24 hours after injection,
but inflammatory cells are not present in large numbers at
14 days, when the tumor is already established (Fig. 2,
top). Apoptotic cells can be found as early as 1 hour (not
shown) and throughout tumor progression (Fig. 2, bottom),
suggesting that the early phases of the inflammatory process
are vital to the host response to the tumor and may define the
tumor outcome.
Among the mediators of the inflammatory response, the metabolites of AA, such as prostaglandins, thromboxanes, and
leukotrienes, have an essential role. In humans, AA is mainly
metabolized by lipoxygenases [including 5-LOX, 12-LOX, and
15-LOX] and cyclooxygenases (including COX-1 and COX-2;
ref. 39). Leukotrienes are molecules derived from the catalytic
action of the 5-LOX enzyme, which generates 5-HpETE and
LTA4. The latter may be further metabolized through the action
of LTA4 hydrolase or leukotriene C4 synthase enzymes, resulting in the production of LTB4 or leukotriene C4, respectively.
5-LOX is expressed in a limited number of cells, mainly leukocytes such as neutrophils, macrophages, mastocytes, and also
by epithelial cells. LTB4 is the main inflammatory mediator
originating from the 5-LOX pathway in neutrophils. This molecule is a potent chemoattractant of neutrophils and macrophages to the inflamed site and can induce angiogenesis (40).
Several studies have shown that 5-LOX or its products, expressed by diverse tumor cell lineages, including melanoma,
may stimulate proliferation and inhibit apoptosis (8, 41). The
role of COX in carcinogenesis has been extensively studied
(42), but little is known about the contribution of 5-LOX to
the inflammatory response associated with many tumors, although a few reports link the 5-LOX metabolite LTA4 (but
not LTB4) to vascular endothelial growth factor expression
and neoangiogenesis (43).
In our model, both COX or 5-LOX inhibitors showed no
influence on Tm1 melanoma proliferation in vitro (data not
shown). However, experiments in vivo showed that the inhibition of 5-LOX pathway (Fig. 3B and C), unlike the inhibition
of the COX pathway (Fig. 3A), was able to reduce melanoma
growth and progression associated with acute inflammation.
This effect was only observed in the coinjection group, with
no effect on those animals injected with a tumorigenic dose
of melanoma cells (2 × 105 cells per animal, control group),
the growth of which is supposedly not related to an inflammatory response (Fig. 3D-F). Interestingly, inhibition of 5-LOX by
caffeic acid modified the timing of neutrophil recruitment to the
injection site, as depicted in Fig. 4A and B. These results suggest that the first few hours in the inflammatory response are
critical for definition of tumor outcome.
LTB4 is the main metabolite derived from the AA/5-LOX
pathway and plays an essential role in leukocyte recruitment
and activation (40). Its expression has been associated with promotion of carcinogenesis (44, 45), tumor progression (46, 47),
and apoptosis resistance (48, 49). Figure 5 confirmed its role in
our model of melanoma progression, while having no effect on
the in vitro growth of Tm1 cell proliferation (Fig. 5B). AA (ω6
PUFA) is not the only membrane lipid metabolized by COX or
LOX enzymes. Diets rich in fish oils contain large quantities of
EPA or docosahexaenoic acid (ω3 PUFAs), and these lipids
may also be metabolized by those enzymes, resulting in the
production of mediators with anti-inflammatory properties
(48). A lower rate of ω6/ω3 PUFA from the diet has been clearly correlated with cancer risk reduction, lower cardiovascular
mortality, and suppression of inflammation in rheumatoid arthritis and asthma (reviewed in ref. 49). Human melanoma incidence is significantly correlated to UV radiation and its
associated inflammatory response (50). Storey et al. (51)
showed that EPA and docosahexaenoic acid were able to reduce interleukin-8, an important chemokine for human neutrophils, in keratinocytes and fibroblasts subject to UV-B
radiation. Injection of EPA or LTB5, the leukotriene from
EPA/5-LOX pathway that corresponds to LTB4, had small
but significant inhibition of melanoma growth associated with
inflammation in our model as well (Fig. 6A and C).
We cannot completely exclude the presence of a smoldering
inflammatory response in our control groups, in which mice
were injected with a full tumorigenic dose of melanoma cells
(2 × 105 Tm1 cells). Nevertheless, the scientific literature at
large does not consider the injection of tumorigenic cells an inflammatory stimulus, in accordance with our own previous
FIGURE 4. Neutrophil infiltrate persists after 5-LOX pathway inhibition.
The inhibition of 5-LOX (caffeic acid; 25 mg/animal/d) does not inhibit the
neutrophil infiltrate in mice submitted to coinjection of apoptotic cells (106
γ-irradiated Tm1) or a subtumorigenic dose of Tm1 (103 viable Tm1 cells;
A, black circles and B, bottom). Subcutaneous tissues from nontreated
(PBS) and treated (caffeic acid) animals were surgically removed 6, 24,
and 72 h after coinjection and processed for immunohistochemistry analysis using a specific antibody against myeloperoxidase, an antigen expressed mainly by neutrophils (×100). *, P < 0.05, statistical significance
using nonpaired nonparametric Student's t tests.
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work (13) that showed no inflammatory infiltrate in the tissue
surrounding the injection site of a large amount of viable melanoma cells.
Melanoma is a notoriously hard disease to manage in later
stages because of an elevated resistance to either chemotherapy
or radiotherapy. Our results show that lipid mediators derived
from the AA/5-LOX metabolism have a fundamental stimulatory role in melanoma progression associated with inflammation, whereas ω3 PUFA–derived metabolites have opposite
effects, and that the early host response to cellular death may
be a determinant of the final outcome of this deadly disease.
Because the same mechanisms that promote progression of a
primary tumor contribute to the establishment of metastatic disease, our findings can be extrapolated to the later phases of
melanoma. Because the current approaches for cancer treatment
(chemotherapy, radiotherapy, and immunotherapy) induce massive apoptosis in the target tissue, these results may have implications in the understanding of cancer treatment failures. The
apoptosis caused by cancer treatment might induce an inflammatory reaction that can promote the proliferation of few surviving cells at the treated site. Rapid tumor relapses observed
after tumor treatment might be related to these mechanisms,
which warrant further investigation.
Merck Sharp and Dohme; LTB4 and LTB4-ethanolamide were
purchased from Cayman Chemical; EPA was purchased from
Calbiochem; and leukotriene B5 was purchased from Sigma.
Materials and Methods
Tumorigenicity Assays
Subconfluent monolayers of Tm1 melanoma cells were harvested after trypsin treatment, counted, and resuspended in culture media at a concentration of 2 × 104 cells/mL. Another
suspension of 2.107 cells/mL was submitted to γ-irradiation
(200 Gy) for induction of apoptosis, using Gammacell 300 Elan
(Nordion Internacional, Inc.). After the irradiation procedure,
both cell suspensions were centrifuged and resuspended in
PBS (140 mmol/L NaCl, 2 mmol/L NaH2 PO 4 , 8 mmol/L
Na2HPO4) and 50 μL of each solution (1 × 103 viable cells plus
1 × 106 apoptotic cells) were admixed and coinjected s.c. in the
flank of syngeneic 6- to 8-wk-old C57Bl/6 female mice. To assess the effect of LPS or carrageenan in tumor progression, animals were coinjected with a mixture of 103 viable Tm1 cells
(50 μL) and LPS (2.5 μg) or carrageenan (0.1%). Animals were
kept under 12-h daylight cycles, without food restriction, and
checked daily for tumor development. Each experimental group
consisted of at least five animals. Tumor growth was monitored
three times per week and tumor volume was determined as follows: [maximum diameter × (minimum diameter)2] / 2 (54).
Mice with a subcutaneous mass >20 mm3 were considered positive for the presence of tumors.
Cell Culture, Reagents, and Proliferation Assay
The murine melanoma cell line Tm1 (52) was cultured in
RPMI (pH 6.9; Gibco) supplemented with 5% FCS (Gibco)
at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
Cell proliferation was determined using a standard MTT assay
(Sigma) following standard protocol (53). LPS and carrageenan
were purchased from Sigma; caffeic acid and MK886 were purchased from Calbiochem; indomethacin was purchased from
Histologic and Immunohistochemistry Analysis
Mice were sacrificed according to institutional guidelines,
and subcutaneous tissues corresponding to the site of coinjection were excised in different periods after injection (6 h, 24 h,
72 h, and 14 d). Samples were submitted to histologic analysis
after paraffin embedding and H&E staining. Hematoxylinstained sections were used for immunohistochemistry analysis
FIGURE 5.
LTB4 is an important
positive mediator of melanoma progression induced by inflammation.
LTB4 (100 nmol/L) is able to promote
the growth of a subtumorigenic dose
of Tm1 melanoma cells (10 3 Tm1
cells; A) but does not promote in vitro
Tm1 proliferation (B). The LTB4 receptor 1 antagonist, LTB4-ethanolamide (Eth; 100 nmol/L/animal/d)
significantly inhibits melanoma tumor
growth in mice coinjected with apoptotic Tm1 melanoma cells (106 γ-irradiated Tm1) and a subtumorigenic
dose of Tm1 (103 viable Tm1 cells;
C), but not in mice injected only with
a tumorigenic dose of Tm1 melanoma cells (2 × 105 cells; D). *, P <
0.05, statistical significance using
nonpaired nonparametric Student's
t tests.
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LTB4 Favors Melanoma Progression
FIGURE 6. Leukotriene B5 pathway
inhibits melanoma progression associated
with an acute inflammatory response.
EPA, a LTB5 precursor, significantly inhibits tumor growth (subtumorigenic dose of
Tm1 melanoma cells; 103 cells) promoted
by an acute inflammatory stimuli (106 γirradiated Tm1; A), but does not abrogate growth of a tumorigenic dose of
Tm1 melanoma cells (2 × 105 cells; B).
Similarly, mice submitted to same coinjection protocol (C), but not to the injection of a tumorigenic dose of melanoma
Tm1 cells ( D), and treated with LTB5
(100 nmol/L) present reduced melanoma
growth. *, P < 0.05, statistical significance using nonpaired nonparametric
Student's t tests.
for neutrophil detection (myeloperoxidase-specific antibody,
Santa Cruz Biotechnology). TUNEL reactions were done on
serial sections using an in situ cell death detection kit (ApopTag
Peroxidase In Situ Apoptosis Detection Kit S7100, Chemicon
International; ref. 55) according to the manufacturer's instructions. Sections were visualized in a Zeiss Axioskop40 microscope, and images were captured using a Sony Cybershot 3.3
megapixels and analyzed in a MetaVue Imaging System v.6.1
(Molecular Devices Corporation) for cell counts.
In vivo Treatment with Modulators of Inflammatory
Pathways
Indomethacin, diluted in sterile PBS, was used at a concentration of 2 mg/kg of weight for each mouse. The treatment program consisted in 10 i.m. doses (100 μL) daily, the first one at
the same time of tumor cell injection. MK886 was used at a
concentration of 1 mg/kg of weight for each mouse, and the
treatment program consisted in six i.p. injections (200 μL) daily, the first at the time of tumor cell injection. Caffeic acid treatment consisted in 10 i.p. injections (2.5 mg/500 μL/animal)
daily, the first one at the moment of injection. For analysis of
the effect of LTB4 in melanoma tumor progression, animals
were s.c. injected with a 100 μL suspension containing 103
Tm1 cells admixed with LTB4 (100 nmol/L final concentration). In addition, a group of animals was treated with LTB4ethanolamide, an antagonist of LTB4 receptor 1 (BLTR1; 100
nmol/L final concentration), at the moment of injection and for
4 more days, in the neighboring injection site through the s.c.
route. EPA (50 μmol/L final concentration) and LTB5 (100
nmol/L final concentration) treatment was similar to LTB4ethanolamide. All experiments were done using groups of five
to ten 6- to 8-wk-old female mice. Control groups received either injections of PBS or a subtumorigenic dose of Tm1 melanoma cells (103) according to the type of experiment (see
Results and figure legends). All doses of anti-inflammatory
agents were based on published studies (56, 57), and the
anti-inflammatory activity of these compounds was confirmed
in vivo using the murine footpad inflammation model.
Statistical Analysis
The data are expressed as the mean ± SD. Nonpaired
nonparametric Student's t test was used to analyze the difference between the means using the InStat software package.
Kaplan-Meier survival analyses by the Mantel-Haenszel logrank test was done by using GraphPad Prism version 4 for
Windows (GraphPad). The significance level was established
at P < 0.05.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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Published OnlineFirst September 8, 2009; DOI: 10.1158/1541-7786.MCR-09-0038
Leukotriene B4 Creates a Favorable Microenvironment for
Murine Melanoma Growth
André Luis Lacerda Bachi, Fabiana Jin Kyung Kim, Suely Nonogaki, et al.
Mol Cancer Res Published OnlineFirst September 8, 2009.
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