Enhanced Inhibitory Effect of Meloxicam, A
Cyclooxygenase-2 Inhibitor Combined with Cisplatin on the Growth of Human
Ovarian Cancers
Bing Xin, Yoshihito
Yokoyama, Tatsuhiko Shigeto, Masayuki Futagami, Hideki Mizunuma
Bing Xin, Yoshihito
Yokoyama, Tatsuhiko Shigeto, Masayuki Futagami, Hideki Mizunuma, Department of Obstetrics and
Gynecology, Hirosaki University Graduate School of Medicine, 5-Zaifu-cho,
Hirosaki, 036-8652 Japan
Bing Xin, Department of Obstetrics and
Gynecology, The Second Affiliated Hospital of China Medical University,
Shengyang, China
Correspondence to: Yoshihito Yokoyama, MD, Department of Obstetrics and Gynecology, Hirosaki University Graduate
School of Medicine, 5-Zaifu-cho, Hirosaki, Aomori 036-8562, Japan.
Email: yokoyama@cc.hirosaki-u.ac.jp
Telephone: +81-172-39-5107
Fax: +81-172-37-6842
Received: September 5, 2013 Revised: October
2, 2013
Accepted: October 8, 2013
Published online: December 18, 2013
ABSTRACT
Cyclooxygenase-2 (COX-2) inhibitors are regarded as potentially important
for cancer treatment and some reports have demonstrated that combination of
COX-2 inhibitors and other anticancer drugs may produce additive or synergistic
activity in the treatment of some human cancers. In this study, we investigated
the effects of combination of meloxicam, a selective COX-2 inhibitor, and
cisplatin (CDDP), a platinum-type drug, on growth of epithelial ovarian cancer
(EOC) cells as well as xenografted tumors derived from EOC cells and evaluated
the ability of this combining treatment on cell proliferation, apoptosis,
prostaglandin E2 level and angiogenesis. While proliferation of HTOA
cells did not change in CDDP alone at a concentration between 0.005-0.5 g/mL, it was significantly decreased by combination
meloxicam and CDDP in a dose-dependent manner of CDDP. Meloxicam alone, CDDP
alone or their combination significantly suppressed the growth of OVCAR-3
tumors xenographted subcutaneously and prolonged the survival of mice with
malignant ascites derived from DISS cells as compared to control. Combination
of meloxicam and CDDP decreased the expression of COX-2, microsomal
prostaglandin E synthase and prostaglandin receptor-3 in tumors. Their
combination significantly decreased prostaglandin E2 and vascular
endothelial growth factor in serum as well as in ascites, and significantly
reduced microvessel density and induced apoptosis in tumors. In conclusion,
these results indicate that combination of meloxicam and CDDP may produce at
least additive antitumor activity in the treatment of EOC. The inhibitory
effect of their combination on growth of EOC suggests a potential to lead a
novel therapeutic strategy against EOC.
© 2013 ACT. All rights reserved.
Key words: Meloxicam; CDDP; Ovarian cancer; PGE2;
Angiogenesis; Apoptosis
Xin B, Yokoyama Y,
Shigeto T, Futagami M, Mizunuma H. Enhanced Inhibitory Effect of Meloxicam, A
Cyclooxygenase-2 Inhibitor Combined with Cisplatin on the Growth of Human
Ovarian Cancers. Journal of Tumor 2013; 1(7): 53-61 Available from: URL:
http://www.ghrnet.org/index.php/JT/article/view/542
INTRODUCTION
Epithelial
ovarian cancer (EOC) represents an insidious disease that typically has
progressed to an advanced stage at the time of diagnosis and no reasonably
sensitive or specific tests exist to make routine screening cost-effective for
early detection or prevention. As such, advances in therapeutic interventions
have had little impact on the long-term reductions in deaths attributable to
EOC. Patients with ovarian cancer have the highest mortality rate among
gynecological malignancies[1] and an estimated 130,000 deaths per
year still occurs worldwide[2]. Duo to the limited success of
current therapy, non-toxic modulators of ovarian cancer growth should be
developed.
The cyclooxygenase-2 (COX-2) protein is highly expressed in
a variety of human cancers including colon, lung and many other solid cancers[3-6].
COX-2 has been associated with tumor growth, angiogenesis, invasion, and
metastasis[7-9]. Overexpression of COX-2 may increase the resistance
of apoptosis in cancer cells[10,11]. The reduction of the COX-2
enzyme activity or protein expression may inhibit the cell survival and growth
in cancer cells. Over the past decade, COX-2 inhibitors have been found to have
chemo-preventive and antitumor activity and to potential the effects of
chemotherapy in variety of tumors[12-14]. The currently proposed
mechanisms of COX inhibitor antitumor activity include: reduction in cell
proliferation, induction of apoptosis, and inhibition of angiogenesis[15,16].
Some evidences prove that COX-2 expression might play an important role in EOC
development[17] and increased COX-2 expression was associated with
chemotherapy resistance and poor outcome in EOC patients[18]. We
proved previously that meloxicam, a selective COX-2 inhibitor produced potent
anti-tumor effect against EOC in conjunction with reduction of angiogenesis and
induction of apoptosis. Recently, COX-2 inhibitors in combination with other
anticancer drugs including doxorubicin, bleomycin, and 5-fluorouracil, have
been evaluated for the anticancer activity in human cancers[19-21].
Selective COX-2 inhibitors enhance the in vitro and in vivo
antitumor effect of anticancer drugs and this effect is associated with
concomitant suppressing intratumor PGE2 levels, altering expression
of Bcl-2, Bax, 15-d PGJ2 and so on.
Cisplatin
(cis-diaminedichloroplatinum (II), CDDP), a clinical anticancer drug, is one of
the platinum-type agents. It exhibits cytotoxic effect due to decreased
proliferation and induction of apoptosis after DNA damage, commonly used in the
treatment of ovarian cancer. The combination of a platinum-type drug and
paclitaxel is standard therapy for the first-line treatment of women with EOC
who require systemic chemotherapy. Recently, cytotoxicity effects of
combination of CDDP with selective COX-2 inhibitors have been evaluated by
different investigators on some type cancer cell lines and proved that
selective cyclooxygenase-2 inhibitors enhanced sensitivity of cancer cells to
CDDP mediated cytotoxicity and apoptosis in vitro and in vivo[22,23].
Combination of COX-2 inhibitors and oxaliplatin, a third-generation platinum,
can increase the growth inhibition and death in human colon cancer cells[24].
In this study, we investigated the
antitumor activity of meloxicam combining with CDDP on growth of EOC in
vitro and in vivo experiments and evaluated the ability of this
combining treatment on regulating prostaglandin E2 levels, apoptosis
and angiogenesis.
METHODS
Cell lines and
cell culture
OVCAR-3 and HTOA
were obtained from the American Type Culture Collection (Rockville, MD) and the
RIKEN Cell Bank (Tsukuga, Japan), respectively. OVCAR-3 was derived from a
patient with poorly differentiated papillary adenocarcinoma of the ovary. HTOA
was established from a well-differentiated human ovarian serous adenocarcinoma.
DISS was kindly provided from Dr. Saga (Jichi Medical School, Tochigi, Japan).
It was derived from human ovarian serous adenocarcinoma. OVCAR-3 and DISS cell
lines grew in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS),
100 U/mL penicillin and 100 g/mL streptomycin, HTOA cell lines grew
in HamF12 medium supplemented with 15% FBS, 100 U/mL penicillin and 100 g/mL streptomycin. All of cell lines were cultured at 37℃ in a water-saturated atmosphere with 5% CO2/95% air. We
checked expression of COX-2 protein with a size of 72 KDa by Western blot
analysis in three cell lines (Figure 1). The bands of COX-2 in Western blot of
these cells disappeared after preincubation of the antibody with a COX-2
peptide (data not shown).
Cell proliferation assay
To study the effects
of meloxicam (Boehringer Ingelheim, Ingelheim, Germany) and CDDP (Nippon
Kayaku, Tokyo, Japan) on proliferation of OVCAR-3, HTOA and DISS cells, 100 L aliquots of cell suspension (5,000 cells/well) in 96-well
microplates were incubated with various concertration of meloxicam or CDDP
alone and their combination for 72 hour. Viable cell number was estimated by
Alamar blue assay and the values were expressed as intensity of fluorescence[25].
Briefly, 10 L of Alamar blue working solution
(BioSource, Camarillo, CA) was added to each well and the plate was further
incubated at 37℃ for 3 hour. The fluorescence intensity
was measured with excitation at 544 nm and emission at 590 nm using a
microplate reader. The reaction was linear in the range of 40-4,000
fluorescence units, corresponding to 5,000-500,000 viable cells/well. Meloxicam
was dissolved in dimethyl sulphoxide (DMSO, Sigma-Aldrich, St. Louis, MO) at a
concentration of 100 M. Drug dilutions were prepared in a culture medium and the DMSO
concentration in the wells with the highest drug concentration did not exceed
1%.
Animal experimentation
The animal
experiments were conducted in accordance with the Guidelines for Animal Experimentation,
Hirosaki University. Eight-week-old female BALB/c nu/nu mice were used in this
study. All mice were group-housed in plastic cages with stainless-steel grid
tops in an air-conditioned and 12 hour light-dark cycle maintained room in the
Institute for Animal Experiments of Hirosaki University and fed with water and
food ad lib. Mice were monitored for health every 3 day and weighed
weekly.
Cancer-bearing mouse model
OVCAR-3 cells (5×106 cells) were inoculated subcutaneously in 500
L of RPMI1640 medium in the back region
of the nude mice. All the mice were numbered, housed separately and examined
twice weekly for tumor development. The tumor was grown until the longer
diameter became 2 mm before starting treatment. Then, the experimental mice
were divided into four groups containing 10 mice each (Day 0). Control group
received basal diet alone. Meloxicam group was given 162 ppm meloxicam in the
diet everyday until the end of the study. CDDP group was administered CDDP at 5
mg/kg intraperitoneally once on Day 0. The combination treatment group was
given meloxicam and CDDP essentially in the same way as administered for their
respective individual treatment regimens. The tumor dimensions were measured
twice weekly using a vernier caliper and tumor volume was calculated using the
equation V (mm3)=A×B2/2, where A is the largest diameter and B is the smallest
diameter[26]. Serum PGE2 concentration was determined on
Day 7, and the mice were sacrificed on Day 21 to remove the tumor for pathological
and biochemical studies.
Cancerous peritonitis mouse model
DISS cells (0.5×107 cells) were inoculated into the peritoneal cavity of the
nude mice in 500 L of sterile PBS. It has been reported
that the average survival of DISS cell-transplanted mice is about 30 days[26].
The experimental mice were divided into four groups of each 8 mice. After
confirming ascites to be produced on Day 7, the mice were treated in the same
way as in the cancer-bearing mouse model. Ascites was aspirated on day 21 for
the determination of PGE2 and VEGF concentrations, and then the
survival time for each group was evaluated.
Western blot analysis
Removed tumor
tissues were cut into small pieces and homogenized in PIPA buffer in ice. The
homogenate was incubated overnight 4℃ followed by centrifuging 15,000g for 15 min at 4℃. The supernatants (50 g protein) were electrophoresed through a 10%
SDS-polyacrylamide gel, and blotted as described previously[27]. The
protein concentration was determined using Bradford’s method. The blots were
probed with the following primary antibodies: COX-2 (IBL, Gunma, Japan) at 2 g/mL or β-actin (Sigma-Aldrich) at 1:2000. The membranes probed by COX-2 were
incubated for 2 hour with anti-rabbit IgG conjugated to horsradish peroxidase
and then visualized the immunoblots using diaminobezidine (DAB) (Sigma-Aldrich)
as a substrate of peroxidase. β-actin was used as a loading control. The membrane probed by β-actin was incubated for 1 hour with biotinylated
anti-mouse immunoglobin, transferred to avidin-biotin-peroxidase complex
reagent (Vector Laboratories, Burlingame, CA), and incubated in this solution
for 30 min. DAB (Sigma-Aldrich) was used as a substrate. Quantification of the
results was performed by scanning the membrane with Photoshop software (version
5.5, Adobe Systems) followed by densitometry with the public domain software,
NIH Image, version 1.62.
Measurement of PGE2 and VEGF
in serum and ascites
PGE2
concentrations were determined with PGE2 EIA system (R and D
Systems, Minneapolis, MN) according to manufacturer’s instructions. VEGF
concentrations were determined using an ELISA kit (R and D Systems) as
described by Gu et al[28].
Immunohistochemical analysis and
microvessel density
Six-m sections of formalin-fixed and paraffin-embedded tissue
specimens were stained by established method as described previously[29].
Sections were incubated with antibodies specific for microsomal PGE synthase
(mPGES) (Cayman Chemical, Ann Arbor, MI), PGE2 receptor-3 (EP3)
(FabGennix, Frisco, TX), or VEGF (R and D Systems) for 1 hour and CD31 (R and D
Systems) overnight. Slides were incubated with biotinylated species-specific
appropriate secondary antibodies for 30 min and then exposed to
avidin-biotin-peroxidase complex (Vector Laboratories). Sections were treated
with 0.02% DAB as a chromogen, and counterstained with hematoxylin. VEGF
expression was evaluated according to a scoring method by the positive cell
percentage and the staining intensity as reported previously[29].
Microvessel density was determined as follows. The highly vascularized areas of
the tumor stained with an anti-CD31 antibody were identified, and CD31-positive
microvessels per 0.75 mm2 were counted under high-power field.
Single endothelial cells or clusters of endothelial cells, with or without a
lumen, were considered to be individual vessels. Microvessel density was
expressed as the vessel number/high-power field in sections. Three fields were
counted per animal, and the average was taken as the microvessel density of
each tumor.
Apoptosis
Apoptosis was
measured on tissue sections by the terminal deoxyribonucleotidyl
transferase-mediated dUTP-biotin nick and labeling assay as described by
Gavrieli et al[30] with some modifications. Briefly, 6-m sections were stripped from proteins by incubation with 10 mg/mL
proteinase K for 15 min and immersed in 0.3% H2O2 in
methanol for 15 min to block the endogenous peroxidase. The sections were then
incubated in TdT mixture buffer (200 mM potassium cacodylate, 25 mM Tris-HCl,
pH 6.5, 0.25 mg/mL BSA, 1 mM CoCl2, 0.01 mM biotin-dUTP, 520 U/mL
TdT) at 37℃ for 1 hour. After rinsed in PBS, the sections were exposed
to avidin-biotin-peroxidase complex at 37℃ for 30 min. Cells undergoing apoptosis were visualized with DAB. The
numbers of stained tumor cells were counted in three fields at ×200 magnification and the results were averaged.
Statistical analysis
The survival curves
were calculated by the Kaplan-Meier method, and the statistical significance of
differences in the cumulative survival curves between the groups was evaluated
by logrank test. Other statistical analyses were carried out by Student’s t-test,
Chi square test or Fisher's exact probability test. A result was deemed
significant at P<0.05. Data are expressed as means±standard deviation
(S.D.)
RESULTS
Inhibitory effect of meloxicam, CDDP
and their combination on cell growth
To examine the
effect of meloxicam and combination of meloxicam and CDDP on in vitro
cell growth, first we exposed three EOC cell lines to meloxicam at a various
concentrations and measured cell proliferation after 72 hours. As shown in
figure 2A, meloxicam inhibited the growth of HTOA cells in at concentrations of
≥100 M or more. On the other hand, although meloxicam did not
inhibit the growth of OVCAR-3 and DISS cells at a concentration of 100 M, meloxicam at concentrations of ≥500 M or more inhibited the growth of both of cell lines.
Next, we assessed the inhibitory
effect of combination of meloxicam and CDDP on proliferation of HTOA cells. A
concentration of meloxicam was fixed at 100 M that reduced 20% of proliferation of HTOA cells, and CDDP
was administered at various concentrations. While cell growth did not change in
CDDP alone at a concentration between 0.005-0.5 g/mL (Figure 2B, white bars), it was significantly
decreased by combination meloxicam and CDDP in a dose-dependent manner of CDDP
(Figure 2B, black bars).
Anti-tumor effect of combination of
meloxicam and CDDP on cancer-bearing mice and cancerous peritonitis mice
To study the
anti-tumor effects of combination of meloxicam and CDDP, we prepared a
cancer-bearing mouse model and a cancerous peritonitis mouse model. In the
cancer-bearing mice, the combination produced a significantly greater antitumor
effect than the control, CDDP, or meloxicam alone treatment. At the end of the
experiment, the tumor volumes were 5.94±0.57, 3.66±0.62, 4.41±0.54, and
2.83±0.62 cm3 in the control, CDDP alone, meloxicam alone, and their
combination, respectively (Table 1). The inhibition rate was 38.4% for CDDP
alone, 25.8% for meloxicam alone, and 52.4% for their combination (Table 1). In
the cancerous peritonitis model, the survival times were significantly prolonged
in CDDP alone, meloxicam alone, and their combination compared with the control
(P<0.0001, respectively, figure 3). The combination significantly
prolonged the survival time as compared with CDDP alone (P<0.05),
whereas no significant difference was found in survival time between meloxicam
alone and the combination (P=0.13) (Figure 3). The combination did not
result in weight loss or gain of the mice and did not lead to any adverse
effects.
Decreased PGE2 levels in
serum and ascites by administration of combination of meloxicam and CDDP
The concentration of
PGE2 in serum of cancer-bearing mice serum was 660±78 pg/mL for the
control group, whereas it was 595±69 pg/mL, 133±31 pg/mL and 104±24 pg/mL for
the CDDP alone, meloxicam alone and their combination, respectively (Figure 3,
white bars). The concentration of PGE2 in serum significantly
decreased in the combination, compared with the CDDP or meloxicam alone (p<0.0001
and p<0.05, respectively). The mean concentration of PGE2
in ascites of cancerous peritonitis mice was 709±256 pg/mL for the control,
whereas it was 505±120 pg/mL, 199±36 pg/mL and 140±45 pg/mL for CDDP alone,
meloxicam alone, and their combination, respectively (Figure 4, black bars).
The concentration of PGE2 in ascites was significantly lower in
their combination than in CDDP or meloxicam alone (p<0.0001 and p<0.01,
respectively). Additionally, the concentration of PGE2 in serum and
ascites significantly decreased in meloxicam alone compared with the control (p<0.0001,
p<0.0001, respectively).
Effect of meloxicam on COX-2 expression
in tumors
Expression of COX-2
in tumors was evaluated by Western blotting. This analysis revealed that in
CDDP alone-treated tumors, COX-2 expression did not change as compared to
control, and that in meloxicam alone-treated tumors and the combination-treated
tumors, the level of COX-2 expression was 56% and 41%, compared to the control,
respectively (Figure 5).
Altered expression of mPGES and EP3 in
tumors by meloxicam or the combination
In order to examine
whether expression of mPGES which converts PGH2 to PGE2,
and EP3 which is one of the PGE2 receptors was altered by
combination of meloxicam and CDDP, distribution of mPGES and EP3 in tumors was
immunohistochemically investigated. We observed stronger staining of mPGES in
the cancer cells in specimens obtained from the control tumors (Figure 6A),
moderate staining in the cancer cells of tumors from CDDP alone (Figure 6B).
There was weak or negligible staining of mPGES in cancer cells of tumors
obtained from meloxicam or the combination (Figure 6C and D). Interestingly, in
meloxicam alone- and the combination-tumors, mPGES appeared stronger staining
of edge cells of cancer nest (Figure 6C and D), whereas in another tumors, its
expression distributed evenly in the peripheral and center of the tumor. The
staining intensity EP3 paralleled to that of mPGES (Figure 6E to H) and its
distribution pattern was almost same as that of mPGES.
Inhibitory effect of combination of
meloxicam and CDDP on VEGF amount in tumors and ascites
VEGF amount in
tumors was evaluated by immunohistochemical staining as shown in figure 7A. The
staining score of VEGF was 5.4±0.5 for the control, 4.3±0.7 for CDDP alone,
2.9±0.9 for meloxicam alone, and 2.2±0.5 for the combination (Figure 7B). The
VEGF amount significantly decreased in the combination as compared to CDDP
alone (P<0.001) or meloxicam alone (P<0.05) alone. VEGF
amount in ascites determined by ELISA were 582.3±112.0 for the control,
391.3±44.8 for CDDP alone, 256.3±43.5 for meloxicam alone, and 215.7±40.7 for
the combination (Figure 7C). The amount significantly decreased in the
combination, compared to CDDP (P<0.01) or meloxicam alone (P<0.05).
Reduction of microvessel density and
induction of apoptosis in tumors by combination of meloxicam and CDDP
We examined the
number of microvessels identified with CD31 in tumors using the immunostaining
method. Microvessel density (MVD) (number/mm2) was 21.2±4.1 for the
control, 19.7±8.0 for CDDP alone, 11.2±2.8 for meloxicam alone, and 7.3±1.5 for
the combination, which significantly decreased in the combination as compared
with the control, CDDP alone and meloxicam alone (P<0.001, P<0.001,
and P<0.05, respectively, Figure 8A). To evaluate the frequency of apoptosis
in tumors, apoptotic cells were stained by the TUNEL method, and TUNEL-positive
cells per 0.75 mm2 were counted in a high power field. The number of
TUNEL-positive cells was 14.2±1.8 for the control, 13.5±2.0 for CDDP alone,
20.3±3.1 for meloxicam alone, and 26.6±4.9 for their combination (Figure 8B).
The incidence of apoptotic cells was significantly higher in the combination
than in the control, CDDP alone, and meloxicam alone (P<0.001, P<0.001,
and P<0.02, respectively, figure 8B).
CONCLUSION
In this study it
emerged that combination of meloxicam and CDDP significantly suppressed the
growth of the solid tumors and peritoneal carcinomatosa derived from human EOC
via reduction of angiogenesis and induction of apoptosis as compared to CDDP
alone or meloxicam alone. These results indicate that their combination may
produce a great antitumor effect on the growth and progress of EOC. Their
combination significantly decreased the PGE2 concentration in serum
and ascites leading to induction of apoptosis and reduction in angiogenesis. This
is the first report describing the inhibitory effect of combination of a COX-2
inhibitor and anti-cancer drug on the growth of ovarian cancer.
The COX-2 protein is
highly expressed in a variety of human cancers and COX-2 inhibitors have been
found to have chemo-preventive and antitumor activity[3-6,12-14].
The antitumor effects of combination of selective COX-2 inhibitors and
anticancer drugs have been intensively evaluated by many investigators in
recent years[19-21]. Although combination of a platinum agent and
taxans has become the standard chemotherapy for EOC, the addition of COX-2
inhibitor to these agents has not been fully evaluated. Accordingly, we
investigated the effects of combination of meloxicam and CDDP on the
development of EOC in in vitro and in vivo.
The present in
vitro experiment showed that meloxicam enhances the in vitro
cytotoxicity of CDDP against EOC cells, suggesting the ability of meloxicam to
enhance a suppressive effect of CDDP on proliferation of EOC cells (Figure 2).
Barnes et al[23] reported that the combination of CDDP and
COX-2 inhibitor increased potentiation of the cytotoxic response in ovarian
carcinoma cells because COX-2 inhibitor blocked PGE2 production. We
also showed that although clofibric acid (CA), PPAR ligand, alone did not affect proliferation of EOC cells,
simultaneous treatment with CDDP and CA significantly suppressed proliferation
of the cells compared with that with CDDP alone. CA reduced PGE2
level in the culture medium, and as a result, the decrease of PGE2
level might enhance the ability of CDDP to induce apoptosis and
anti-angiogenesis[31]. Thus, because meloxicam significantly
decreased PGE2 concentration as shown in figure 4, enhancement of
anti-proliferative and anti-tumor effects of CDDP on EOC might result from
reduction in PGE2 level by meloxicam (Figure 2, 3, and table 1). It
has been shown that PGE2 enhances angiogenesis through the induction
of VEGF[26] and represses apoptosis by maintaining Bc1-2 expression[32].
Munkarah et al also reported that in vitro PGE2
treatment stimulated proliferation of ovarian cancer cells and reduced
apoptosis[33]. PGE2 production is involved in the ability
of cancer cells to invade, metastasize, and grow[34]. Our data
suggest that the pronounced tumor growth inhibition associated with the
combination treatment is paralleled by the greatest inhibition of PGE2
levels indicating the important role of PGE2 in the tumor
development.
Although there was
significant difference in reduction of COX-2 expression in tumors between
meloxicam alone and combination of meloxicam and CDDP, their combination
significantly reduced PGE2 concentration in serum and ascites as
compared to meloxicam alone. So, we investigated whether reduction of PGE2
concentration is associated with alteration in the expression of other enzymes
involved in the PGE2 biosynthesis and activation. The biosynthesis
of PGE2 from arachidonic acid requires two enzymes that act
sequentially. COX is a time-limiting enzyme in the synthesis of PGE2
by converting arachidonic acid to PGG2 and subsequently to PGH2,
and mPGES converts COX-derived PGH2 to PGE2. The mPGES
also plays an important role in releasing PGE2 from cancer cells[35]
and its enhanced expression is important in tumorigenesis. Cells overexpressing
both of COX-2 and mPGES produced more PGE2, grew faster and
exhibited more aberrant morphology than those expressing either COX-2 or mPGES
alone[36]. Moreover, PGE2 exerts biological function via
connecting with its membrane-bound receptors. There are at least four
membrane-bound receptors, EP1-4, liganding with PGE2. Out of those,
EP3 is most close to activation of PGE2 and is overexpressed in
human tumor tissues[37]. Rask et al have reported that the
expression of COX-2, mPGE synthase and PGE2 receptor is increased in
EOC and that PGE2-synthesis and signaling are important for
malignant transformation and progression of EOC[38]. The present
immunohistochemical staining showed that staining for mPGES and EP3 in the
combination-treated tumors was weaker than in the control (Figure 6). These
results indicate that alterations in the expression of mPGES and EP3 cause the
reduction of PGE2 level.
Angiogenic factors and
anti-apoptotic factors are downstream targets of PGE2, and as
increase in PGE2 results in up-regulation of VEGF as well as
suppression of apoptosis[39]. Angiogenesis plays a crucial role in
tumor development and progression. PGE2 is a potent inducer of
angiogenesis in vivo and induces expression of angiogenic regulatory
proteins such as VEGF[40]. Naruse et al reported that
meloxicam markedly reduced the expression of VEGF in lung metastatic lesion
from osteosarcoma compared to the control tissues[41]. Celecoxib, a
COX-2 inhibitor could suppress growth of lung and breast tumors by its potent
antiangiogenic activity from reduction of COX-2-derived PGE2[42,43].
The present study revealed that combination of meloxicam and CDDP in vivo
significantly reduced VEGF levels in tumors and the malignant ascites (Figure
7) as well as MVD in tumors (Figure 8A) as compared to meloxicam or CDDP alone,
and that their combination significantly produced apoptotic cells in tumors as
compared to meloxicam or CDDP alone (Figure 8B). It is suggested in this study
that reduction in VEGF and MVD and induction of apoptosis may be via PGE2
reduction by combination of meloxicam and CDDP.
In conclusion, the present results
suggest that combination of meloxicam and CDDP enhances the in vitro and
in vivo antitumor effect via suppression of PGE2
concentration and activation. Their combination inhibited the growth of EOC
through reducing angiogenesis and inducing apoptosis. This combination may
provide a new strategy in human EOC and should be clinical tested.
ACKNOWLEDGMENTS
This study was
supported in part by a Grant-in Aid for Cancer Research (No. 20591935) from the
Ministry of Education, Science and Culture of Japan and by the Karoji Memorial
Fund of the Hirosaki University Graduate School of Medicine
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Peer reviewers: Kaei Nasu, MD, PhD, Division of Obstetrics and Gynecology,
Department of Regional Medicine, Faculty of Medicine, Oita University,
Idaigaoka 1-1, Hasama-machi, Yufu-shi, Oita 879-5593, Japan.
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