ORIGINAL ARTICLE

Cysteinyl Leukotriene Receptor Antagonists Inhibit Tumor
Metastasis by Inhibiting Capillary Permeability

Masako Nozaki,1, 2, 3 Masanobu Yoshikawa,1 Kunihiko Ishitani,2 Hiroyuki Kobayashi,1, 3 Kiyohiro Houkin,4Kohzoh Imai,5 Yoichiro Ito3, 6 and Takamura Muraki3, 7

Department of Clinical Pharmacology, Tokai University School of Medicine, Kanagawa, Japan
2
Higashi Sapporo Hospital, Sapporo, Japan
3
Inflammation Research Center Co. Ltd., Sapporo, Japan
4
Department of Neurosurgery, Sapporo Medical University School of Medicine, Sapporo, Japan
5
Sapporo Medical University School of Medicine, Sapporo, Japan
6
Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute,
National Institutes of Health, Maryland, USA
7
Department of Pharmacology, Tokyo Women’s Medical University School of Medicine, Tokyo, Japan
1(Received for publication on August 25, 2009)(Revised for publication on October 31, 2009)(Accepted for publication on November 5, 2009)

We explored the possibility of the cysteinyl leukotriene receptor antagonists, pranlukast andmontelukast, preventing tumor cell migration through both cerebral and peripheral capillaries.
To study tumor cell migration through brain capillaries, male Fisher rats were cannulated via
the cisterna magna under pentobarbital anesthesia. RCN9 cells labeled with a fluorescentmarker PKH67 were intravenously administered following arachidonic acid administration
into the subarachnoid space, and specimens of the central nervous system were collected every 30 min for 8 h. Arachidonic acid increased the fluid volume with elevated white blood celland RCN9 cell counts. When given 2 h before arachidonic acid administration, pranlukast, but
not montelukast, reduced the fluid volume and inhibited white blood cell and RCN9 cell extravasation through the brain capillary. In addition, a Lewis lung carcinoma metastasis modelin mice was used to study the inhibitory effect of pranlukast and montelukast against cancer
cell extravasation through general capillaries. When mice were given food containing either
pranlukast or montelukast, immediately after paw amputation, tumor metastasis was prevented by both drugs, and their survival was prolonged. These results show that pranlukast can inhibit tumor cell migration through both the brain and peripheral capillaries, whereas montelukast inhibits tumor cell migration only in the peripheral capillaries. (Keio J Med 59 (1) : 10－18,
March 2010)

Keywords: pranlukast, montelukast, cysteinyl leukotriene receptor antagonist, metastasis inhibition,
capillary permeability

Introduction

tastasis. Since it has been reported that the adhesion ofcancer cells to vascular endothelial cells is a key step intumor cell extravasation,1, 2 many studies have focusedon the inhibition of the binding of sialyl Lewis x and sialyl Lewis a antigen with E- and P-selectins on the activated endothelium by using competitive ligand inhibitors, metabolic carbohydrate mimetics, or neutralizing

Metastasis is the primary cause of death in most cancer
patients. Nevertheless, an anti-tumor metastasis agent is
not yet available for clinical use.
It is known that selectins, integrins, cadherins, andCD44 play an important role in hematogenic tumor me-

Reprint requests to: Masako Nozaki, PhD, Department of Clinical Pharmacology, Tokai University School of Medicine, 143 Simokasuya, Isehara, Kanagawa
259-1193, Japan, E-mail: tokai-irc@tokai-u.jp10Keio J Med 2010; 59 (1): 10－ 18

11

monoclonal antibodies. However, these efforts have not
yet produced any fruitful results. Kannagi3 suggested
that the inhibition of E-, P-, and L-selectin might blocktumor cell extravasation.
Cysteinyl leukotriene receptor antagonists such as
pranlukast, montelukast, and zafirlukast are well known
inhibitors of slow-reacting substance of anaphylaxis(SRSA), a mixture of LTC4, LTD4, and LTE4, and hence,
useful for treating asthma and allergy by suppressing
both capillary permeability and white blood cell (WBC)extravasation. This fact led us to consider that these cysteinyl leukotriene receptor antagonists might also inhibit
tumor metastasis because cell extravasation is a common
event in inflammation and tumor metastasis.
In our present study, 2 animal models are proposed toinvestigate cell extravasation during the process of inflammation and tumor metastasis in rats and mice. In
vivo morphological studies revealed useful informationon the process of tumor cell extravasation. Using theseanimal models, our studies were mainly focused on demonstrating the anti-metastatic action of 2 leukotriene receptor antagonists, i.e., pranlukast and montelukast. Possible clinical applications of these drugs are also discussed.

PKH67 Green Fluorescent Cell Linker Kit, a product of
Zynaxis Cell Science Inc., were purchased from Dainippon Sumitomo Pharmaceutical Co. (Osaka, Japan). Arachidonic acid (sodium salts) from porcine liver was obtained from Calbiochem (San Diego, CA, USA). Dextran(M.W. 190,000–230,000) was obtained from Nacalai
Tesque Inc. (Kyoto, Japan), and 2-methylbutane was obtained from Kishida Chemical Co., Ltd. (Osaka, Japan).
Malinol, a mounting agent, was obtained from Muto
Pure Chemicals Co., Ltd. (Tokyo, Japan). Tissue-Tek
OCT compound was obtained from Sakura Finetek (Tokyo, Japan). Sterile phosphate buffered saline (PBS),
which contains 144 mg (=1.06 mM) of KH2PO4, 9,000
mg (=154 mM) of NaCl, and 795 mg (=5.60 mM) of Na2
HPO4 in 1,000 ml of H2O was obtained from Cambrex
Charles City Inc. (IA, USA). ONON, containing 112.5
mg/capsules of pranlukast, was purchased from Ono
Pharmaceutical Co. (Osaka, Japan). Singulair tablets (10mg) that contain 10 mg/tablet of montelukast and are a
product of Merck & Co. Inc. (White House Station, NJ,
USA) were purchased from Banyu Pharmaceutical Co.,
Ltd. (Tokyo, Japan).

Materials and Methods

A cryostat (Tissue Tek Cryo 2,000; Bayer Co., NY,
USA), fluorescence microscopes (ProvisAX x 80; Olympus, Tokyo, Japan and Axiovert S 100; Zeiss, Germany),
and a stereotaxic unit (Type SRS-6; Narishige Scientific
Instrument Lab, Tokyo, Japan) were used.

Tumor cell lines and culture conditions
A rat colon cancer cell line (RCN9) was obtained from
Riken BRC Cell Bank, Tsukuba, Japan, and a mouse
Lewis lung carcinoma (LLC) cell line was purchasedfrom Health Science Research Resources Bank, Osaka,
Japan. Cells were cultured at 37ºC under 5% CO 2 in
PRMI-1640 (SIGMA, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine(Gibco, Grand Island, NY, USA), 100 units penicillin,and 100 μg/ml streptomycin (Gibco).
Animals
Male Fisher 344 rats (weighing approximately 400 ±50g) and female BDF1 mice (6 weeks old) were purchased from Sankyo Lab-Service Co. (Sapporo, Japan).
Animals were maintained in a ventilated, temperaturecontrolled (24 ± 1Cº) envelopment, on a bed of woodshavings with free access to water and food and were
subjected to a 12-h light-dark cycle. The present experiments were in compliance with the guidelines for animalcare followed at the Sapporo Medical University Schoolof Medicine.
Chemicals
Sodium pentobarbital (Nembutal injection), a productof Abbott Laboratories (Abbott Park, Chicago, IL) and

Instruments

Miscellany
Polyethylene tube (SP-10; I.D.: 0.28 mm; O.D.: 0.61mm) was obtained from Natsume Seisakusho Co. (Tokyo, Japan).
The morphological clarification of tumor cell extravasation in vivo
1) RCN9 cell extravasation through the brain capillary
Rats were anesthetized using sodium pentobarbital (50mg/kg i.p.). A femoral vein was exposed for the intravenous injection of cancer cells. The rats were then placedin a stereotaxic unit. An 18-gauge needle was inserted
through the dura via the cisterna magna, and a cannula(SP-10) was introduced into the subarachnoid space(Fig. 1A). The cannula was fixed with cyanoacrylic glue
to the surrounding peripheral tissue. Arachidonic acid(816 ng/2 μl of PBS) was administered through the cannula, and subsequently, specimens of the CNS (CNS
specimens) were collected.
RCN9 cells that originated from the Fisher rats were
stained with a fluorescent marker PKH 67 linker kit.
These cells (between 1.88 × 104 and 2.43 × 104) weresuspended in PBS, which amounts to approximately

Nozaki M, et al: Pranlukast Inhibits Tumor Metastasis

12

A

Fig. 1 An SP-10 polyethylene tube inserted into rat cisterna magna was used for administration of arachidonic acid and collection of
cerebrospinal specimens (A). Capillary of pia mater was observed by fluoromicroscopy 30 min after intracerebrospinal injection ofarachidonic acid in rats administered without (B) RCN9 cells. Arrowheads show the endothelial cell junction (B).

1/100th of the number of neutrophils in the body of eachrat, were intravenously administered immediately before
arachidonic acid injection to the subarachnoid space.
RCN9 cells in the CNS specimen were examined undera fluorescent microscope (Axisovert S 100), and the cellswith a flat, square shape were counted.
2) RCN9 cell extravasation in the general capillary
RCN9 cells were intravenously administered immediately before intradermal injection of 0.1 ml of 0.01%
dextran into the rat’s hind paw; this induces edema without WBC extravasation. The fresh tissues of the paw
were harvested 15–30 min after dextran injection andcarefully embedded into OCT compound in a plastic
mold, while taking care to avoid trapping air bubbles.
The mold was covered with a piece of flat metal, soakedin 2-methylbutane, and frozen with liquid nitrogen for 30
seconds. Then, 10-μm-thick frozen sections were obtained using the cryostat, mounted on the slides, dried on
the heating plate for 30 seconds and finally fixed with amounting agent (malinol). These frozen sections of therat paw were examined for the presence of RCN9 cells
under a fluorescent microscope (ProvisAX x 80).
The pharmacological studies evaluating cysteinyl leukotriene receptor antagonists in the arachidonic acid-induced inflammation and tumor metastasis
Pranlukast (450 mg/kg) or montelukast (10 mg/kg)
was administered orally 2 h before arachidonic acid injection into the subarachnoidal space. Equipotent dosesof each cysteinyl leukotriene receptor antagonist werechosen taking into account the daily dose recommended
for clinical use. RCN9 cells were intravenously administered immediately before arachidonic acid injection. A
CNS specimen was collected every 30 min after arachidonic acid injection and the volume of CNS specimen,

WBC counts, and RCN9 cell counts were measured for aperiod of 8 h.
Effect of cysteinyl leukotriene receptor antagonists on aspontaneous metastasis model of LLC cells
LLC cells (5 × 105) were inoculated into the footpad of
BDF1 mice for a passage culture. When the diameter oftumors reached 10 mm, the tumor-cell-inoculated footwas amputated under ether anesthesia. Then, the mice
were given powdered food containing pranlukast (0 mg/
kg, 7.5 mg/kg, 41.25 mg/kg, 225 mg/kg, 450 mg/kg, and675 mg/kg) or montelukast (0 mg/kg, 0.178 mg/kg, and10 mg/kg). These doses were adjusted mg/kg/day bychanging the drug concentration in the powdered food.
The survival rate was evaluated 4 months after amputation and used as an indicator for anti-metastatic effect ofthese leukotriene receptor antagonists on peripheral
spontaneous metastasis.
Statistical analysis
All data were summarized, and we analyzed the total
volume of the CNS specimen and counts of WBC and
RCN9 cells collected over an 8-h experimental period.
Comparison between the means of multiple groups wereanalyzed by the Mann-Whitney’s U-test using SPSS
16.0J for Windows (SPSS Inc. Chicago, IL, USA) in
pharmacological studies for evaluating cysteinyl leukotriene receptor antagonists on arachidonic acid-induced
inflammation and tumor metastasis.
Effect of cysteinyl leukotriene receptor antagonists on
a spontaneous metastasis model of LLC cells was analyzed by plotting Kaplan-Meier survival curves4 with
log-rank and Wilcoxon test using PRISM ver.5 software
package (GraphPad Software, Inc., La Jolla, CA, USA).
Significance was set at P < 0.05.

Keio J Med 2010; 59 (1): 10－ 18

13

Fig. 2 Images of the process of RCN9 extravasations (A and B) and their schematic presentation (C). Contour of RCN9 cells is traced (A).

Results
Morphological studies
1) RCN9 cell extravasation through the brain capillary
The morphological changes in the brain capillaries
were studied by intravenous injection of the fluorescentmarker solution free of RCN9 cells and subsequent injection of arachidonic acid into the subarachnoidal space(Fig. 1B). A piece of the brain capillary was obtained in
the CNS specimen within 30 min of arachidonic acid injection. Arrowheads pointing towards the irregular gaps
indicate the openings of the blood brain barrier causedby arachidonic acid. Arachidonic acid induced RCN9cell extravasation in the brain capillary (Fig. 2A and
2B). Figure 2C was the schematic presentation for theimage of Figure 2A. The process of transmigration ofcancer cells from the brain capillaries was revealed morphologically by comparing the abovementioned images.
The outlines of cancer cells at the inside and the outsideof the brain capillary are traced by the dotted and thesolid lines, respectively (Fig. 2A), on the original picture(Fig. 2B). RCN9 cells (a and b) in Figure 2A and 2C areshown escaping from the brain capillary. RCN9 cell c in
Figure 2A and 2C has extravasated. As observed in the
CNS specimen, the RCN9 cell after extravasation is flatand square shaped. Dark square-shaped substances

shown with arrowheads in Figure 2A are neutrophils adhered to the endothelial cells. Figures 3A and 3B are
representative images of RCN9 cells obtained within 30
min after extravasations from the brain capillary in the
CNS specimen.
2) RCN9 cell extravasation through the general capillary
The morphological changes of cancer cells were examined in the general capillary in the rat paw edema induced by dextran. Figure 4A shows RCN9 cells attachedto the inside of the peripheral capillary. Arrowheads
show RCN9 cells that were adhered to the gaps of endothelial cells. The shape of the RCN9 cells had changed
from spherical to flat and square before transmigration
through the junctions of endothelial cells. Figure 4B
shows RCN9 cells (shown by arrowheads) during thetransmigration through the general capillary; however,neither white blood cell adhesion nor the transmigrationin the peripheral capillary is induced by dextran (Fig. 4A
and 4B).
The pharmacological studies evaluating cysteinyl leukotriene receptor antagonists in increased capillary permeability and WBC extravasation in the inflammation induced by arachidonic acidArachidonic acid (816 ng/2 μl) injection into the sub-

Nozaki M, et al: Pranlukast Inhibits Tumor Metastasis

14

Fig. 3 Representative images of RCN9 cells (A and B) in the CNS specimen within 30 min after extravasations.

Fig. 4 Histological section of dextran-induced edema in rat foot pad. Arrowheads show RCN9 cells. The general capillary is shownwith (A) or without orange color filter (B).

arachnoidal space immediately increased capillary permeability, which continued throughout an 8-h experimental period (Fig. 5A). On the other hand, WBC counts
started to increase 30–60 min after arachidonic acid injection, maintaining its high level until the end of the experimental period (Fig. 5A). Remarkably, pranlukast inhibited both increased capillary permeability (Fig. 5B
and 6A) and WBC extravasation (Fig. 5B and 6B) induced by arachidonic acid in the brain capillary, whereasmontelukast exerted no such inhibitory effect under otherwise identical experimental conditions (Fig. 5C), instead, it caused an enhancement of both capillary permeability and WBC extravasation (Fig. 6A)
The effect of cysteinyl leukotriene receptor antagonists
on RCN9 cell extravasation induced by arachidonic acid
When RCN9 cells were intravenously injected immediately before arachidonic acid to the subarachnoidal
space, RCN9 cell extravasation was observed in the CNS
specimen immediately after arachidonic acid injection(Fig. 5D). The RCN9 cell counts peaked at 30 min after
arachidonic acid injection and then decreased sharply(Fig. 5D). Both, increased capillary permeability and
RCN9 cell extravasation were inhibited by pretreatment
with pranlukast (450 mg/kg) (Fig. 5E and 6A), but notwith montelukast (10 mg/kg) (data not shown).
The effect of leukotriene receptor antagonist on a spontaneous metastasis model of Lewis lung carcinoma cells
The lung metastasis experiment was performed to determine the survival rate of mice for up to 4 months after
paw amputation. Lung cancer metastasis was proven bygross examination of the lung after the death of mice. All
the mice that did not receive the cysteinyl leukotriene receptor antagonist died within 1 month. The survival ratesof mice treated with pranlukast (7.5–675 mg/kg) (Fig.
7A and Table 1) and montelukast (0.178–10 mg/kg)(Fig. 7B and Table 1) treatments were as high as 66–
100%, although they were not dose dependent.
Discussion
A quantitative method to determine increased capillarypermeability of the brain microvasculature and extravasation of both WBCs and tumor cells is described in thispaper. This method includes subarachnoidal injection ofarachidonic acid followed by collection of CNS effluent
from the cisterna magna. We assume that the CSF effluent consists mainly of inflammatory exudates from thebrain capillary. It provides reliable data because collect-Keio J Med 2010; 59 (1): 10－ 18

15

Fig. 5 Time-dependent effect of pranlukast and montelukast on the arachidonic acid-induced CNS specimen (μl) (◇), counts of WBC(■) (A, B, C), and RCN9 cells (�) (D, E) are shown. Symbols represent the mean and the s.e.m. n = 6. Rats were pretreated withpranlukast (B, E), montelukast (C), or distilled water (A, D), and CNS specimen was collected every 30 min up to 8 h after arachidonic
acid injection.

ing CNS specimens from the same set of animals at given time intervals can eliminate statistical variations that
could arise while sampling between individual animals.
This method was first applied to determine the phase thatis inhibited by drugs during the process of inflammationand tumor metastasis. Furthermore, this method can beused as a tool to clarify the mode of action of the anti-inflammatory and/or anti-tumor metastatic drugs.
Our in vivo morphological studies on both the brain
and general capillary revealed that cancer cells can passthrough narrow gaps in between the capillary endothelial
cells by flattening themselves. The ability of cancer cells

to change their shape from spherical to flat and square
may facilitate transmigration through the gap between
endothelial cells shortly after arachidonic acid administration; the transmigration of WBCs had a delay of over
30 min. The fact that cancer cell extravasation started
immediately after a noxious stimulus such as arachidonic
acid injection suggests the possibility of causing acutemetastasis directly through the capillary rather thanforming emboli as generally believed. Therefore, it may
be necessary for cancer patients to start taking cysteinyl
leukotriene receptor antagonist drugs before the surgery
to inhibit this form of acute metastasis.

16

Nozaki M, et al: Pranlukast Inhibits Tumor Metastasis

Fig. 6 The summary of the results of pharmacological effects of cysteinyl leukotriene receptor antagonists on inflammation and tumor
cell migration induced by arachidonic acid in the brain capillary. Total volume of cerebrospinal specimen (A) and counts of WBC (B)and RCN9 cells (C) examined over an 8-h experimental period are calculated from Figure 3(A–E). Bars represent the mean and thes.e.m. n = 6.

Fig. 7 Effect of pranlukast (A) and montelukast (B) on the Kaplan-Meier survival curve of a spontaneous metastasis model in mousefor 4 months after amputation of the paw inoculated with Lewis Lung Carcinoma cells. Log rank test and Wilcoxon test are shown as
inset. n = 6–12.

Our morphological studies do not focus on the effectof cysteinyl leukotriene receptor antagonists on dextraninduced cancer cell extravasation since dextran-induced
paw edema is not recognized as an inflammation becauseof a lack of WBC extravasation. The only reason of using dextran-induced edema is to demonstrate how cancer
cells change their shape during transmigration throughthe general capillary without the effects of other factors
such as WBC extravasation.
This is the first report showed that pranlukast, but notmontelukast, is able to inhibit increased capillary permeability and extravasation of both WBCs and tumor cells
through the brain capillary. As the blood brain barrier has
continuous tight junctions, drug penetration into thebrain must depend on transcellular rather than paracellular transport. Therefore, it is necessary to overcome thislimitation of the blood brain barrier to deliver anti-in-

flammatory agents and/or anti-tumor metastasis agentsthat otherwise do not cross the blood brain barrier, andfor this reason, there is no drug available for the clinicaltreatment of encephalic inflammation and brain tumor.5
Drugs that open the blood brain barrier would cause extravasation of blood components from the brain capillary
to the CNS because of the large difference between
blood pressure (80–140 mm Hg) and intracranial pressure (7–15 mm Hg), thereby giving rise to adverse physical conditions in the patients. Since cysteinyl leukotriene
receptors are present at the outside of the brain capillary,cysteinyl leukotriene receptor antagonists that do not, or
only minimally, cross the blood brain barrier 6 shouldhave no effect on the brain capillary. In fact, the present
study demonstrated that montelukast, which is known as
a pure cysteinyl leukotriene receptor antagonist, failed toinhibit the extravasation of both WBCs and cancer cells

Keio J Med 2010; 59 (1): 10－ 18

17

Table 1 Summary of statistics of Kaplan-Meier survival curves
dose(mg/kg)
pranlukast
7.50
41.25
225.00450.00675.00montelukast
0.175
10.00

log rank test

Gehan-Breslow Wilcoxon testp < 0.0001
p < 0.0001
p < 0.0001
p < 0.0001
p < 0.0001

p < 0.0001
p < 0.0006
p < 0.0006
p < 0.0006
p < 0.0001

p < 0.0001
p < 0.0001

p < 0.0001
p < 0.0001

in the brain capillary. The results of our study suggestthat arachidonic acid-induced capillary permeability
might be further increased by pretreatment with montelukast because of an unknown mechanism. This unexpected finding should be confirmed by future studies.
The inhibitory effect of pranlukast against both inflammation and tumor metastasis through the brain capillary
might be attributed to some particular mode of action via
peripheral and/or the interior of the blood vessel, including the endothelial cells and the capillary lumen. Pranlukast might block VEGF7, 8 and/or glycoprotein in the
blood vessels, because over 98.9% of pranlukast binds toplasma proteins after oral administration in rats.9 Therefore, it is possible that pranlukast inhibits not only tumor
metastasis but also tumor growth.
Because of the effect of leukotriene receptor antagonists on a spontaneous metastasis model of the LLC
cells, both montelukast and pranlukast treatments produced increased survival rates, suggesting that these leukotriene receptor antagonists may inhibit peripheral tumor metastasis. Since the inhibitory effect of pranlukast
and montelukast on the permeability in the general capillary have been well documented and used for the treatment of asthma and allergy,10-16 the inhibitory effect on
the general capillary was not tested in the present study.
Hoff, et al. reported that the adhesion of cancer cells tovascular endothelial cells is considered to be a key stepin tumor cell extravasation,1, 2 Kobayashi, et al.1 7 clarified that metastasis is inhibited by the prevention of cancer cell adhesion to the endothelial cells because of theblocking E-selectin expression, and Kannagi3 proposedthat inhibiting the expression of P-, E-, and L-selectins
induces a blockade of metastasis. We speculate that pranlukast suppresses expression of all selectins on the vascular endothelial cells, thereby inhibiting tumor cell adhesion and extravasation.
While the process of tumor metastasis and inflammation, once started, is progressive and consecutive, enhanced capillary permeability is a prerequisite, enabling
cell extravasation through the capillary. Hence, suppressing the capillary permeability with pranlukast is considered to be the most effective means of preventing thesepathological events. Because the mediators that increase

capillary permeability are same to those that activate Eand P-selectin on the vascular endothelial cells pranlukast might block not only leukotriene receptor but also
the mediators.
Cimetidine was first reported as an anti-tumor metastatic drug in 1979.18 Matsumoto19 reported that cimetidine had a beneficial effect on survival in colorectal cancer patients, the inhibitory effect of cimetidine (100 mg/
kg administered intramuscularly) on increased capillarypermeability was studied using our rat model and wasfound to be potent. It suggests that cimetidine might beuseful in inhibiting acute metastasis during surgeries forcolorectal cancer patients. Although no severe adverse
effects were described in their study, long-term use of cimetidine has, in general, adverse side effects, such as inhibition of P450 system, headache, and gynecomastia. In
contrast pranlukast has long been used for treating asthma chronically with little adverse effects.20
The clinical applications of pranlukast, as suggested by
the present study, include prevention of systemic tumor
metastasis and treatment of brain edema, secondary injuries in cerebral circulation failure, and systemic inflammation such as sepsis.
Acknowledgements
We wish to thank Dr. Tomoko Sonoda, Ph.D., for her
statistical analysis and Mr. Mikio Fukuda for drawing
the figures.
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