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Research Articles: Therapeutics, Targets, and Development
Clofibric acid, a peroxisome proliferator–activated receptor
ligand, inhibits growth of human ovarian cancer
1 Department of Obstetrics and Gynecology, and 2 Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki, Aomori, Japan; and 3 Department of Pathology, Faculty of Medicine, Kuwait University, Safat, Kuwait
Requests for reprints: Yoshihito Yokoyama, Department of Obstetrics and Gynecology, Hirosaki University School of Medicine, 5-Zaifu-cho, Hirosaki, 036-8562, Japan. Phone: 81-172-39-5107; Fax: 81-172-37-6842. E-mail: yokoyama{at}cc.hirosaki-u.ac.jp
Abstract
Recent reports have shown that peroxisome proliferator–activated receptor (PPAR)
ligands reduce growth of some types of malignant tumors and prevent carcinogenesis. In this study, we investigated the inhibitory effect of clofibric acid (CA), a ligand for PPAR on growth of ovarian malignancy, in in vivo and in vitro experiments using OVCAR-3 and DISS cells derived from human ovarian cancer and aimed to elucidate the molecular mechanism of its antitumor effect. CA treatment significantly suppressed the growth of OVCAR-3 tumors xenotransplanted s.c. and significantly prolonged the survival of mice with malignant ascites derived from DISS cells as compared with control. CA also dose-dependently inhibited cell proliferation of cultured cell lines. CA treatment increased the expression of carbonyl reductase (CR), which promotes the conversion of prostaglandin E2 (PGE2) to PGF2, in implanted OVCAR-3 tumors as well as cultured cells. CA treatment decreased PGE2 level as well as vascular endothelial growth factor (VEGF) amount in both of OVCAR-3–tumor and DISS-derived ascites. Reduced microvessel density and induced apoptosis were found in solid OVCAR-3 tumors treated by CA. Transfection of CR expression vector into mouse ovarian cancer cells showed significant reduction of PGE2 level as well as VEGF expression. These results indicate that CA produces potent antitumor effects against ovarian cancer in conjunction with a reduction of angiogenesis and induction of apoptosis. We conclude that CA could be an effective agent in ovarian cancer and should be tested alone and in combination with other anticancer drugs. [Mol Cancer Ther 2007;6(4):1379–86]
Introduction
The current management of advanced epithelial ovarian cancer generally includes cytoreductive surgery followed by combination chemotherapy. The combination of paclitaxel with a platinum analogue is the preferred chemotherapy regimen in the treatment of newly diagnosed patients with this disease (1). Although such management induced the favorable results in the treatment of ovarian cancer, the long-term survival of ovarian cancer patients remains unsatisfactory (2), and an estimated 130,000 deaths per year still occur from ovarian cancer worldwide (3). Acquired drug tolerance of the ovarian cancer cells is regarded as one of the causes that fail to prolong the survival period of the ovarian cancer patients (4). Although it is important to elucidate the mechanisms of overcoming drug resistance, new medication, apart from the known chemotherapeutic agents, remains to be developed with a view to improving survival and cure rates in ovarian cancer.
Peroxisome proliferator–activated receptors (PPAR) belonging to the nuclear hormone receptor superfamily exist as three isoforms: PPAR, PPARß/
, and PPAR
. PPAR plays physiologic roles in fatty acid metabolism and catabolism, and PPAR regulates adipocyte differentiation (5), although functions of PPARß/ are not fully known. Several reports show that PPAR is implicated in carcinogenesis, and ligands for PPAR inhibit the growth of human breast, prostate, gastric, and lung cancer (6–9). However, because there has been significant interest in the role of PPAR in metabolic disorders, data on PPAR and its ligands in human malignancies are scant.
Clofibric acid (CA) is a ligand for PPAR and is clinically used for the treatment of hyperlipidemic disorders. CA treatment causes a transcriptional increase of acyl CoA oxidase (ACO) and bifunctional enzyme (BE; enoyl CoA hydratase/L-3-hydroxyacyl CoA dehydrogenase), which are peroxisomal enzymes involved in fatty acid ß-oxidation, in the liver of rats via PPAR activation (10, 11). The expression of several genes coding for fatty-acid–metabolizing enzymes including ACO are elevated in response to PPAR ligands and are often used as markers of PPAR activation (12).
A recent report showed that a selective ligand for PPAR could exert its protective role against mouse skin tumor promotion (13). It has also more recently emerged that PPAR is up-regulated in endometrial cancer, and a PPAR-activating ligand reduces the proliferation of endometrial cancer cells (14). These findings raise a great interest in the association between PPAR and ovarian malignancy because epidemiologic data suggest that dietary fat and cholesterol may increase the risk of ovarian cancer by increasing circulating estrogen levels (15). There have been, however, no data on PPAR and its ligands in ovarian cancer. In this study, we investigated the effect of CA on the growth of malignant ovarian tumors in in vivo and in vitro experiments and aimed to elucidate the molecular mechanism of its antitumor effect. Here, we used cisplatin [cis-diaminedichloroplatinum(II) (CDDP)], which is a DNA-damaging agent used in the chemotherapy of human malignancy and is considered one of the key drugs for ovarian cancer treatment, as a positive control of the antitumor effect in order that the effect of CA could be compared with that of CDDP.
Materials and Methods
Cell Lines and Cell Culture
OVCAR-3 was obtained from the American Type Culture Collection (Rockville, MD), and DISS was kindly provided from Dr. Saga (Jichi Medical School, Tochigi, Japan). OVCAR-3 cells have been commonly used to produce xenografted solid tumor (16), and the xenograft of DISS cells to the peritoneal cavity of mice has been reported to produce peritonitis carcinomatosa (17). Both cell lines were derived from human ovarian cancers, grown in RPMI 1640 supplemented with 10% FCS, 100 units/mL penicillin, and 100 µg/mL streptomycin at 37°C in a water-saturated atmosphere with 5% CO2/95% air.
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-h–light-dark–cycle–maintained room in the Institute for Animal Experiments of Hirosaki University and fed with water and food ad libitum.
Cancer-Bearing Mouse Model
OVCAR-3 cells (0.5 x 107 cells) were inoculated s.c. in 0.2 mL of RPMI 1640 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 six mice each (day 0). The control group received basal diet alone. The CA group was given 9,000 ppm CA (Sigma-Aldrich, St. Louis, MO) in the diet everyday until the end of the study. The CDDP group was given CDDP (Nippon Kayaku, Tokyo, Japan) at 5 mg/kg i.p. once on day 0. The combination treatment group was given CA and CDDP essentially in the same way as administered for their respective treatment regimens. The tumor dimensions were measured twice weekly using a caliper, and tumor volume was calculated using the equation V (mm3) = A x B2/2, where A is the largest diameter and B is the smallest diameter (18). Serum prostaglandin E2 (PGE2) concentration was determined on day 7, and the mice were sacrificed on day 21 to remove the tumor for pathologic and biochemical studies.
Cancerous Peritonitis Mouse Model
DISS cells (0.5 x 107 cells) were inoculated into the peritoneal cavity of the nude mice in 0.5 mL of sterile PBS. It has been reported that the average survival of DISS cell-transplanted mice is about 30 days (17). The experimental mice were divided into four groups of eight mice each. 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 were aspirated on day 21 for the determination of PGE2 and vascular endothelial growth factor (VEGF) concentrations, and then the survival time for each group was evaluated.
Apoptosis
Apoptosis was measured on tissue sections by the terminal deoxyribonucleotidyl transferase (TdT)–mediated dUTP-biotin nick end labeling (TUNEL) assay as described by Gavrieli et al. (19), with some modifications. Briefly, 6-µm 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 mmol/L potassium cacodylate, 25 mmol/L Tris-HCl (pH, 6.5), 0.25 mg/mL bovine serum albumin, 1 mmol/L CoCl2, 0.01 mmol/L biotin-dUTP, 520 units/mL TdT] at 37°C for 1 h. After rinsing in PBS, the sections were exposed to avidin-biotin-peroxidase complex (VECTA Laboratories, Burlingame, CA) at 37°C for 30 min. Cells undergoing apoptosis were visualized with 3,3'-diaminobenzidine (DAB; Sigma-Aldrich). The numbers of stained tumor cells were counted in three fields at x200 magnification, and the results were averaged.
Immunohistochemical Analysis and Microvessel Density
Six-micrometer sections of formalin-fixed and paraffin-embedded tissue specimens were stained by the established method as described previously (20). Sections were incubated with antibodies specific for carbonyl reductase (CR; ref. 11), VEGF (R&D Systems, Minneapolis, MN), and CD31 (R&D Systems) for 2 h. Slides were incubated with biotinylated species–specific appropriate secondary antibodies for 30 min and then exposed to avidin-biotin-peroxidase complex. 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 (20). 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 a high-power field. 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.
Western Blot Analysis
Cell lysates (50 µg protein) were prepared from cultured cells and tumor tissues, electrophoresed through a 12.5% SDS-polyacrylamide gel, and blotted as described previously (21). The protein concentration was determined using Bradford's method. The blots were probed with the following diluted antibodies for 2 h: CR and BE (10) at 1:1,000, PPAR (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:200, VEGF (R&D Systems) at 1:500, microsomal PGE synthase (mPGES; Cayman Chemical, Ann Arbor, MI) at 1:1,000, and ß-actin (Sigma-Aldrich) at 1:2,000. The membranes were then incubated for 1 h with the appropriate biotinylated secondary antibodies, transferred to avidin-biotin-peroxidase complex reagent, and incubated in this solution for 30 min. DAB was used as a substrate. Quantification of the results was done 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&D Systems) according to manufacturer's instructions. VEGF concentrations were determined using an ELISA kit (R&D Systems) as described by Gu et al. (22).
Cell Proliferation Assay
To study the effect of CA on the proliferation of OVCAR-3 and DISS cells, 90-µL aliquots of cell suspension (5,000 cells per well) in 96-well microplates were incubated with 0, 0.5, 5, 50, or 500 µmol/L CA for 72 h. Viable cell number was estimated by Alamar blue assay, and the values were expressed as intensity of fluorescence (23). Briefly, 10 µL of Alamar blue working solution (BioSource, Camarillo, CA) was added to each well, and the plate was further incubated at 37°C for 2 h. 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 to 4,000 fluorescence units, corresponding to 5,000 to 500,000 viable cells per well.
Transfections
T-Ag-MOSE, which was established through the introduction of SV40 large T antigen DNA into C3H/He mouse ovarian surface epithelium and could be transfected cDNA with great efficiency (24), was obtained from Health Science Research Resources Bank (Osaka, Japan). T-Ag-MOSE cells were plated in 24-well plates and cultured until 60% to 80% confluency in DMEM containing 10% FCS. Full-length cDNA coding for mouse CR 1 was ligated into the pBabe Puro expression vector to generate CR expression vector (25). About 10 µg of the coding plasmid was transfected into T-Ag-MOSE cells, using GenePORTER transfection reagent (Gene Therapy Systems, San Diego, CA) in accordance with the manufacturer's recommendations. The vector without CR1 cDNA was used as the control. Transfected cells were cultured in DMEM with 10% FCS containing 1.5 mg/mL puromycin for 48 h. We confirmed the expression of the CR construct using Western blotting.
Tissue Samples
Fresh surgical specimens of epithelial ovarian cancer were obtained from 70 patients who were treated at the Hirosaki University Hospital after informed consent had been obtained. Tumor specimens were fixed in 10% formaldehyde and embedded in paraffin for immunohistochemistry. No patients had received preoperative chemotherapy. All the patients received postoperative chemotherapy, combining CDDP, epirubicin, and cyclophosphamide.
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 log-rank test. Other statistical analyses were carried out by Student's t test, 
2 test, or Fisher's exact probability test. A result was deemed significant at P < 0.05.
Results
Antitumor Effect of CA in Cancer-Bearing Mouse Model and Cancerous Peritonitis Mouse Model
To study the in vivo antitumor effect of CA, we prepared a cancer-bearing mouse model and a cancerous peritonitis mouse model. In the cancer-bearing mouse model, tumor volumes were significantly reduced from day 7 in the CA and CDDP groups, compared with the control group (Fig. 1A
), and the same trend lasted until they were sacrificed on the third week. In the comparison of final tumor weights, tumor inhibition rate was 34% for the CDDP group and 46% for the CA group, with tumors being significantly smaller in each treatment group than in the control group (Fig. 1B). The tumor-reducing effects were similar in the CA and CDDP groups. In the cancerous peritonitis model, the survival times were significantly prolonged in the CA and CDDP groups, compared with the control group (Fig. 1C). No significant difference in survival time was observed between the CDDP and CA groups.
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. To examine whether CR is involved in the decrease of PGE2 following CA treatment, CR expression was evaluated by Western blotting. This analysis revealed that CR expression in tumors increased after CA treatment (Fig. 5A
). Then, to examine whether CA induces CR expression in cultured cells, CA was added to the culture medium of OVCAR-3 cells at concentrations of 0 to 500 µmol/L, and after 48 h culture, it was found that CR expression increased depending on the dose of CA (Fig. 5B). PPAR as well as BE expression also increased dose dependently (Fig. 5B). CA treatment repressed PGE2 concentration in culture medium, from 172.0 ± 65.0 to 21.0 ± 9.8 ng/mL at a 500 µmol/L CA concentration. The value was not significantly altered, or CR was not induced by CDDP treatment. Then, to study the effect of CA on in vitro cell growth, CA was added to OVCAR-3 and DISS cells. After 72 h culture, cell growth was determined using Alamar blue assay. CA inhibited growth dose dependently in both types of cells (Fig. 5C; P < 0.05, respectively). Our previous in vitro experiment using rat hepatocytes showed that a 500 µmol/L CA concentration induced peroxisomal enzymes such as ACO and BE without any toxicities (10).
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In this study, it emerged that CA suppressed the growth of solid and peritonitis ovarian malignancies derived from human ovarian cancer cells in conjunction with the reduction of angiogenesis and significant induction of apoptosis. CA also inhibited cell proliferation dose dependently in cultured OVCAR-3 and DISS ovarian cancer cells.
Although there has been significant interest in the role of PPAR in metabolic disorders, there are few reports on PPAR in human malignant diseases, much less in ovarian cancer. Holland et al. (14) showed that up-regulation of PPAR was one of the transcript changes identified in endometrial cancer using cDNA microarray, and that treatment with fenofibrate, a ligand for PPAR, significantly reduced proliferation and increased cell death in cultured endometrial cancer cells expressing PPAR. Braissant et al. (26) found expression of PPAR in rat ovaries using in situ hybridization. Toda et al. (27) reported that fenofibrate inhibited aromatase cytochrome P450 expression in the ovary of mouse, but did not in PPAR null mice, indicating an important function of PPAR in the ovaries. Genes induced by PPAR are primarily associated with fatty acid ß-oxidation in the cells (28). Although the possibility has been reported that the PPAR ligands could reduce growth of some types of malignant tumors (14, 29) and prevent carcinogenesis (13), the mechanism remains unsettled. In this study, it was found that expression of not only CR but also BE involved in fatty acid ß-oxidation increased in the ovarian cancer cells depending on the dose of CA (Fig. 5B), suggesting that PPAR is present in ovarian cancer cells and activated by CA.
It has been shown that PGE2 enhances angiogenesis through the induction of VEGF (18) and represses apoptosis by maintaining Bc1-2 expression (30). Munkarah et al. (31) also reported that in vitro PGE2 treatment stimulated proliferation of ovarian cancer cells and reduced apoptosis. PGE2 is involved in the ability of cancer cells to invade, metastasize, and grow (32). CR is a cytoplasmic enzyme expressed in the lung, liver, kidney, and ovary, and its main function is to reduce carbonyl compounds with NADPH (33). CR specifically promotes conversion of PGE2 to PGF2 (34). Thus, it may be an important design against tumor growth to try to enhance intracellular expression of CR, which can decrease PGE2 level. It emerged in this study that CA treatment induced CR expression in both ovarian xenografted tumors and in vitro ovarian cancer cells. Furthermore, we obtained the results that high expression of CR decreased the amount of PGE2 and lowered VEGF expression through the transfection experiment as shown in Fig. 6. On the other hand, mPGES, which promotes the conversion of PGH2 to PGE2, plays an important role in releasing PGE2 from a lung cancer cell line (35). It has been shown that a selective cyclooxygenase-2 inhibitor repressed the expression of mPGES in the tumor and reduced intratumor PGE2 level (36). Similarly, we found in this study that CA treatment reduced the expression of mPGES in OVCAR-3 tumors, resulting in a decrease of PGE2 level.
Thus, the mechanism of the antitumor effect of CA may involve two pathways: a pathway through which CA increases intracellular CR level that converts PGE2 into PGF2 and another where CA decreases mPGES, which converts PGH2 to PGE2 in the arachidonic acid cascade, resulting in decreased PGE2 synthesis. The reduced PGE2 levels through these pathways repress VEGF expression, resulting in the inhibition of angiogenesis and also induce apoptosis in tumor cells.
We previously reported a novel function for CR, namely, its ability to modulate the metastatic potential of malignant mouse cells (25). Transfection of the low metastatic-potential subline, derived from mouse lung adenocarcinoma cells, with a construct expressing antisense carbonyl reductase rendered the cells highly metastatic, and conversely, transfection of the high metastatic-potential subline with a construct expressing sense carbonyl reductase decreased their metastatic capacity (25). Furthermore, we found that the decrease or loss of CR expression in epithelial ovarian cancer significantly correlated with retroperitoneal lymph node metastasis and poor survival (20). The present findings suggest that a decrease of PGE2 and a down-regulation of VEGF expression may be involved in the repression of metastasis by CR expression.
Additionally, combined treatment with CA and CDDP showed a greater antitumor effect on ovarian cancer in this study (data not shown). The intrinsic nature of CDDP as a chemotherapeutic agent is the interruption of DNA synthesis and the subsequent cell division in cancer cells (37). Thus, the mechanism of the antitumor effect of CDDP evidently differs from that of CA. The antitumor effect, by combination of both in this study, might just be additive through their different mechanisms. In the meantime, CR is known to reduce the anthracycline anticancer drug, doxorubicin, to doxorubicinol, which is the major metabolite, and might play a key role in the development of doxorubicin-induced cardiotoxicity (38). Inhibition of the toxic effects of CR in the heart has been reported to improve the use of doxorubicin in chemotherapy (39).
It also emerged that CR had a significant inverse correlation with VEGF in its expression pattern using surgical specimens of ovarian cancer (Table 1). In this case, it is expected that if CR expression is increased in any way, it can decrease PGE2 amount and VEGF expression, resulting in the inhibition of angiogenesis. Activation of other PPAR isoforms, PPAR and PPARß/, also correlates with the prevention of tumor growth (40, 41). However, their ligands did not activate CR at all, and CA treatment did not affect the expression of PPAR and PPARß/ in in vitro experiment using OVCAR-3 cells (data not shown). CA is clinically used as a well-tolerated hyperlipidemic agent, with the most common adverse events related to the digestive system. We conclude that CA could be an effective agent in human ovarian cancer and should be tested alone and in combination with other molecular-targeted agents or cytotoxic drugs.
Footnotes
Grant support: Grant-in Aid for Cancer Research (16591632) from the Ministry of Education, Science and Culture of Japan and the Karoji Memorial Fund of the Hirosaki University School of Medicine.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11/22/06; revised 1/23/07; accepted 2/22/07.
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  D. Panigrahy, A. Kaipainen, S. Huang, C. E. Butterfield, C. M. Barnes, M. Fannon, A. M. Laforme, D. M. Chaponis, J. Folkman, and M. W. Kieran PPAR{alpha} agonist fenofibrate suppresses tumor growth through direct and indirect angiogenesis inhibition PNAS, January 22, 2008; 105(3): 985 - 990. [Abstract] [Full Text] [PDF]
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