Enhanced Inhibitory Effect of Meloxicam, A Cyclooxygenase-2 Inhibitor Combined with Cisplatin on the Growth of Human Ovarian Cancers

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

 

 

REFERENCES

1    Greenlee RT, Murray T, Bolden S, Wingo PA. Cancer statistics. Ca Cancer J Clin 2000; 50(1):  7-33

2    Shibuya K, Mathers CD, Boschi-Pinto C, Lopez AD, Murray CJ. Global and regional estimates of cancer mortality and incidence by site: II. Results for the global burden of disease 2000. BMC Cancer 2002; 2: 37

3    Dixon DA, Tolley ND, King PH, Nabors LB, McIntyre TM, Zimmerman GA, Prescott SM. Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells. J Clin Invest 2001; 108(11): 1657-1665

4    Brabender J, Park J, Metzger R, Schneider PM, Lord RV, Hölscher AH, Danenberg KD, Danenberg PV. Prognostic significance of cyclooxygenase 2 mRNA expression in non-small cell lung cancer. Ann Surg 2002; 235(3): 440-443

5    Shariat SF, Kim JH, Ayala GE, Kho K, Wheeler TM, Lerner SP. Cyclooxygenase-2 is highly expressed in carcinoma in situ and T1 transitional cell carcinoma of the bladder. J Urol 2003; 169(3): 938–942

6    Chen WS, Liu JH, Wei SJ, Liu JM, Hong CY, Yang WK. Colon cancer cells with high invasive potential are susceptible to induction of apoptosis by a selective COX-2 inhibitor. Cancer Sci 2003; 94(3): 253–258

7    Tabernero A, Reimund JM, Chasserot S, Muller CD, Andriantsitohaina R. Cyclooxygenase-2 expression and role of vasoconstrictor prostanoids in small mesenteric arteries from patients with Crohn's disease. Circulation 2003; 107(9): 1407–1410

8    Chen WS, Wei SJ, Liu JM, Hsiao M, Kou-Lin J, Yang WK. Tumor invasiveness and liver metastasis of colon cancer cells correlated with cyclooxygenase-2 (COX-2) expression and inhibited by a COX-2-selective inhibitor, etodolac. Int J Cancer 2001; 91(6): 894–899

9    Han C, Leng J, Demetris AJ, Wu T. Cyclooxygenase-2 promotes human cholangiocarcinoma growth: evidence for cyclooxygenase-2-independent mechanism in celecoxib-mediated induction of p21waf1/cip1 and p27kip1 and cell cycle arrest. Cancer Res 2004; 64(4): 1369–1376

10   Krysan K, Merchant FH, Zhu L, Dohadwala M, Luo J, Lin Y, Heuze-Vourc'h N, Põld M, Seligson D, Chia D, Goodglick L, Wang H, Strieter R, Sharma S, Dubinett S. COX-2-dependent stabilization of survivin in non-small cell lung cancer. FASEB J 2004; 18(1): 206–208

11   Krysan K, Dalwadi H, Sharma S, Põld M, Dubinett S. Cyclooxygenase 2-dependent expression of survivin is critical for apoptosis resistance in non-small cell lung cancer. Cancer Res 2004; 64(18): 6359–6362

12   Lynch PM. COX-2 inhibition in clinical cancer prevention. Oncology 2001; 15(Suppl.5): 21-26

13   Reddy BS, Hirose Y, Lubet R, Steele V, Kelloff G, Paulson S, Seibert K, Rao CV. Chemoprevention of colon cancer by specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages of carcinogenesis. Cancer Res 2000; 60(2): 293-297

14   Soriano AF, Helfrich B, Chan DC, Heasley LE, Bunn PA Jr, Chou TC. Synergistic effects of new chemopreventive agents and conventional cytotoxic agents against human lung cancer cell lines. Cancer Res 1999; 59(24): 6178-6184

15   Dempk W, Rie C, Grothey A, Schmoll HJ. Cyclooxygenase-2: a novel target for cancer chemotherapy? J Cancer Res Clin Oncol 2001; 127(7): 411-417

16   Mohammed SI, Bennett PF, Craig BA, Glickman NW, Mutsaers AJ, Snyder PW, Widmer WR, DeGortari AE, Bonney PL, Knapp DW. Effects of the cyclooxygenase inhibitor, piroxicam, ontumor response, apoptosis, and angiogenesis in a canine model of human invasive urinary bladder cancer. Cancer Res 2002; 62(2): 338-340

17   Shigemasa K, Tian X, Gu L, Shiroyama Y, Nagai N, Ohama K. Expression of cyclooxygenase-2 and its relationship to p53 accumulation in ovarian adenocarcinomas. Int J Oncol 2003; 22(1): 99-105

18   Ferrandina G, Lauriola L, Zannoni GF, Fagotti A, Fanfani F, Legge F, Maggiano N, Gessi M, Mancuso S, Ranelletti FO, Scambia G. Increased cyclooxygenase-2 (COX-2) expression is associated with chemotherapy resistance and outcome in ovarian cancer patients. Ann Oncol 2002; 13(8): 1205-1211

19   Hashitani S, Urade M, Nishimura N, Maeda T, Takaoka K, Noguchi K, Sakurai K. Apoptosis induction and enhancement of cytotoxicity of anticancer drugs by celecoxib, a selective cyclooxygenase-2 inhibitor, in human head and neck carcinoma cell lines. Int J Oncol 2003; 23(3): 665-672

20   Chen WS, Liu JH, Liu JM, Lin JK. Sequence-dependent effect of a cyclooxygenase-2 inhibitor on topoisomerase I inhibitor and 5-fluorouracil -induced cytotoxicity of colon cancer cells. Anticancer Drugs 2004; 15(3): 287-294

21   Shaik MS, Chatterjee A, Jackson T, Singh M. Enhancement of antitumor activity of docetaxel by celecoxib in lung tumors. Int J Cancer 2006; 118(2): 396-404

22   Mizutani Y, Nakanishi H, Li YN, Sato N, Kawauchi A, Miki T. Enhanced sensitivity of bladder cancer cells to cisplatin mediated cytotoxicity and apoptosis in vitro and in vivo by the selective cyclooxygenase-2 inhibitor JTE-522. J Urol 2004; 172(4): 1474-1479

23   Barnes AP, Miller BE, Kucera G. Cyclooxygenase inhibition and hyperthermia for the potentiation of the cytotoxic response in ovarian cancer cells. Gynecol Oncol 2007; 104(2): 443-450

24   Lin J, Hsiao PW, Chiu TH, Chao JI. Combination of cyclooxygenase-2 inhibitors and oxaliplarin increases the growth inhibition and death in human colon cancer cells. Biochem Pharmacol 2005; 70(5): 658-667

25   Ahmed SA, Gogal RM Jr, Walsh JE. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. J Immunol Methods 1994; 170(2): 211-224

26   Yokoyama Y, Xin B, Shigeto T, Umemoto M, Kasai-Sakamoto A, Futagami M, Tsuchida S, Al-Mulla F, Mizunuma H. Clofibric acid, a peroxisome proliferator-activated receptor ligand, inhibits growth of human ovarian cancer. Mol Cancer Ther 2007; 6(4): 1379-1386

27   Sakamoto A, Yokoyama Y, Umemoto M, Futagami M, Sakamoto T, Bing X, Mizunuma H. Clinical implication of expression of cyclooxygenase-2 and peroxisome proliferator activated-receptor in epithelial ovarian tumours. Br J Cancer 2004; 91(4): 633-638

28   Gu JW, Elam J, Sartin A, Li W, Roach R, Adair TH. Moderate levels of ethanol induce expression of vascular endothelial growth factor and stimulate angiogenesis. Am J Physiol Regul Integr Comp Physiol 2001; 281(1): R365-372

29   Umemoto M, Yokoyama Y, Sato S, Tsuchida S, Al-Mulla F, Saito Y. Carbonyl reductase as a significant predictor of survival and lymph node metastasis in epithelial ovarian cancer. Br J Cancer 2001; 85(7): 1032-1036

30   Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119(3): 493-501

31   Yokoyama Y, Xin B, Shigeto T, Futagami M, Mizunuma H. Clofibric acid, a peroxisome proliferator–activated receptor alpha ligand, enhances a suppressive effect of cis-diaminedichloroplatinum on proliferation of ovarian carcinoma cells. Clin Med Insights Oncol 2007; 1(1): 1-6

32   Sheng H, Shao J, Morrow JD, Beauchamp RD, DuBois RN. Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 1998; 58(2): 362-366

33   Munkarah AR, Morris R, Baumann P, Deppe G, Malone J, Diamond MP, Saed GM. Effects of prostaglandin E2 in proliferation and apoptosis of epithelial ovarian cancer cells. J Soc Gynecol Investig 2002; 9(3): 168-173

34   Yang VW, Shields JM, Hamilton SR, Spannhake EW, Hubbard WC, Hylind LM, Robinson CR, Giardiello FM. Size-dependent increase in prostanoid levels in adenomas of patients with familial adenomatous polyposis. Cancer Res 1998; 58(8): 1750-1753

35   Catley MC, Chivers JE, Cambridge LM, Holden N, Slater DM, Staples KJ, Bergmann MW, Loser P, Barnes PJ, Newton R. IL-1-dependent activation of NF-B mediates PGE2 release via the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase. FEBS Lett 2003; 547(1-3): 75-79

36   Murakami M, Naraba H, Tanioka T, Semmyo N, Nakatani Y, Kojima F, Ikeda T, Fueki M, Ueno A, Oh S, Kudo I. Regulation of prostaglandin E2 biosynthesis by inducible membraneassociated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2. J Biol Chem 2000; 275(42): 32783–32792

37   Amano H, Hayashi I, Endo H, Kitasato H, Yamashina S, Maruyama T, Kobayashi M, Satoh K, Narita M, Sugimoto Y, Murata T, Yoshimura H, Narumiya S, Majima M. Host Prostaglandin E2-EP3 Signaling Regulates Tumor-Associated Angiogenesis and Tumor Growth. J Exp Med 2003; 197(2): 221-232

38   Rask K, Zhu Y, Wang W, Hedin L, Sundfeldt K. Ovarian epithelial cancer: a role for PGE2-synthesis and signalling in malignant transformation and progression. Mol Cancer 2006; 5: 62

39   Wang D, Dubois RN. Prostaglandins and cancer. Gut 2006; 55(1): 115-122

40   Chang SH, Liu CH, Conway R, Han DK, Nithipatikom K, Trifan OC, Lane TF, Hla T. Role of prostaglandin E2-dependent angiogenic swich in cyclooxygenase 2-induced breast cancer progression. Proc Natl Acad Sci USA 2004; 101(2): 591-596

41   Naruse T, Nishida Y, Hosono K, Ishiguro N. Meloxicam inhibits osteosarcoma growth, invasiveness and metastasis by COX-2-dependent and independent routs. Carcinogenesis 2006; 27(3): 584-592

42   Fulzele SV, Shaik MS, Chatterjee A, Singh M. Anti-cancer effect of celecoxib and aerosolized docetaxel against human non-small cell lung cancer cell line, A549. J Pham Pharmacol 2006; 58(3): 327-363

43   Basu GD, Pathangey LB, Tinder TL, Lagioia M, Gendler SJ, Mukherjee P. Cyclooxygenase-2 inhibitor induces apoptosis in breast cancer cells in an in vivo model of spontaneous metastatic breast cancer. Mol Cancer Res 2004; 2(11): 632-642

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.

 

Refbacks

  • There are currently no refbacks.