Abstract
The purpose of this study was to elucidate the possible mechanism that clofibric acid (CA), a peroxisome proliferator-activated receptor α ligand, enhances a suppressive effect of cis-diaminedichloroplatinum (CDDP) on proliferation of the ovarian carcinoma line OVCAR-3. These cells was incubated with 0.5 μM/ml CDDP in the presence or absence of 50 μM CA or incubated with 50 μM CA alone for 72 hr. While treatment with CA alone did not affect proliferation of OVCAR-3 cells, simultaneous treatment with CDDP and CA significantly suppressed proliferation of the cells and significantly induced apoptosis compared to that with CDDP alone. Treatment with CDDP and CA significantly decreased the prostaglandin (PG) E2 level in the medium of the cells compared with treatment with CDDP alone. These results suggest the ability of CA to enhance a suppressive effect of CDDP on proliferation of the ovarian carcinoma cells through reduction of PGE2 level.
Introduction
Epithelial ovarian carcinoma 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 of this disease. The current management of advanced epithelial ovarian carcinoma generally includes cytoreductive surgery followed by combination chemotherapy, using platinum combined with a taxan such as paclitaxel. Although such management induced the favorable results in the treatment of ovarian carcinoma, the long-term survival of ovarian carcinoma patients remains unsatisfactory (Yokoyama et al. 1999). Approximately 70% of all initial responders to chemotherapy relapse and subsequently require additional therapy as second- or third-line chemotherapy (Muggia, 2004). Acquired drug tolerance of the ovarian carcinoma cells is regarded as one of the causes that fail to prolong the survival period of the ovarian carcinoma patients. In order to overcome drug resistance or maintain sensitivity to chemotherapeutic agents, the development of new chemotherapeutic agents, addition of translational agents or the devices of the administration of the agents should be required. Also, the mechanisms for enhancement of sensitivity to anticancer agents remain to be elucidated.
Cisplatin (cis-diaminedichloroplatinum (II), CDDP) is a DNA-damaging agent that is widely used in the chemotherapy of human malignancies and is considered one of the key drugs for ovarian carcinoma treatment (Cohen and Lippard, 2001). Chemotherapeutic effect of CDDP relies primarily on its ability to induce apoptosis in tumor cells (Ormerod et al. 1994). Resistance to CDDP is not completely understood although it is postulated that it would occur through many different mechanisms. Up-regulation of cyclooxygenase (COX) enzymes in ovarian tumor cells has been implicated to play a key role in platinum drug resistance (Munkarah and Ali-Fehmi, 2005). Enhanced COX-2 gene expression is thought to be associated with CDDP resistance and the promotion of tumor progression (Munkarah et al. 2002; Sakamoto et al. 2004). COX-2 produces PGE2 in cell membrane primarily by converting arachidonic acid to prostaglandin (PG) G2 and secondly by converting PGG2 to PGH2.
Peroxisome proliferator-activated receptors (PPARs) belong to a superfamily of intracellular ligand-activated receptors (Isseman and Green, 1990). Currently three subtypes of mammalian PPARs have been identified, termed α, β and γ, encoded by different genes and showing distinct tissue distribution (Sorensen et al. 1998). PPARα acts as a regulator of lipid metabolism and plays a crucial role in liver metabolism (Schoonjans et al. 1996). Clofibric acid (CA), a hypolipidemic agent, is known to be one of PPARα ligands (Peters et al. 2003). We previously reported that addition of CA to the cultured hepatocytes increased mRNA expression and activity of acyl CoA oxidase and bifunctional enzyme (enoyl CoA hydratase/L-3-hydroxyacyl CoA dehydrogenase), key enzymes of peroxisomal β-oxidation system (Yokoyama et al. 1993). Furthermore, addition of CA reduced transcriptional activity of c-jun and resulted in loss of mRNA expression of glutathione S-transferase (GST) π form which acts as detoxification of xenobiotics such as anticancer agents (Yokoyama et al. 1993). Taking these results together, we supposed that CA could augment the sensitivity of carcinoma cells to anticancer agents via decreased expression of GST-π. However, CA did not affect expression of GST-π and other multi-drug resistance-related proteins such as p-glycoprotein and multidrug resistance associated protein in the ovarian carcinoma cells (Yokoyama et al. 1998).
We conducted a preliminary study of whether CDDP could suppress proliferation of ovarian cancer cells in the presence of CA and found the capacity of CA to enhance a suppressive effect of CDDP on their proliferation. The purpose of this study was to elucidate the possible mechanism for the enhanced antiproliferative effect of CDDP by CA.
Materials and Methods
Cell culture
Ovarian carcinoma cell line, OVCAR-3 was obtained from the American Type Culture Collection (Rockville, U.S.A). Cells were grown in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a water-saturated atmosphere with 5% CO2/95% air.
Cell proliferation assay
To study the effect of CA on proliferation of OVCAR-3 cells, 90 μl aliquots of cell suspension (5,000 cells/well) in 96-well microplates were incubated with 0.5 μM/ml CDDP in the presence or absence of 50 μM CA or incubated with 50 μM CA alone for 72 hr. We preliminarily confirmed that addition of 0 to 50 μM CA to OVCAR-3 cells did not affect cell proliferation (Yokoyama et al. 2007). As a negative control, the cells were cultured in a medium without CDDP and CA. Viable cell number was estimated by Alamar blue assay and the values were expressed as intensity of fluorescence (Ahmed et al. 1994). Briefly, 10 μl of Alamar blue working solution (BioSource, Camarillo, USA) was added to each well and the plate was further incubated at 37 °C for 2 hr. The fluorescence intensity was measured with excitation at 544 nm and emission at 590 nm using a BIORAD Model 550 microplate reader (Bio-Rad Laboratories, Tokyo, Japan). The reaction was linear in the range of 40–4,000 fluorescence units, corresponding to 5,000–500,000 viable cells/well.
Morphological change of apoptosis
Cells were seeded at 5 × 10 5 cells per 10 cm-diameter dish in growth medium. Twenty-four hours later, the medium was replaced by the fresh medium (10% FBS) containing 50 μM CA, 0.5 μM/ml CDDP or 0.5 μM/ml CDDP + 50 μM CA. Cells were then incubated for 72 hr. As a negative control, the cells were cultured in growth medium without any agents. Briefly, the experimental cells were fixed in 4% paraformaldehyde for 30 min at room temperature (RT) and then washed once with PBS. Fifty ng/ml of Hoechst 33258 (Dojin, Kumamoto, Japan) was added to the fixed cells, incubated for 30 min at RT, and washed with phosphate buffer saline (PBS). The cells were mounted and visualized under a fluorescence microscope. Apoptotic cells were identified by the condensation and fragmentation of their nuclei. The percentage of apoptotic cells was calculated from the ratio of apoptotic cells to total cells counted. More than 500 cells were counted for each treatment. There was not any non-specific staining.
Determination of PGE2 concentration
The PGE2 concentration in the supernatant of the cultured cells was quantified by a commercial high sensitivity PGE2 enzyme immunoassay kit (Neogen corporation, Lexington, U.S.A) in reference to the PGE2 standard. Cells were seeded at 5,000 cells per well in microplates in the growth medium. Twenty hours later, the medium was replaced by the serum free fresh medium containing 50 μM CA, 0.5 μM/ml CDDP or 0.5 μM/ml CDDP + 50 μM CA. Cells were then incubated for 24 hr. The supernatants were aspirated and centrifuged to prepare for the detection of PGE2. After incubation at 4 °C for 18 to 24 hr, the excess reagents were washed away and substrate was added. After incubation at 37 °C for 1 hr, the enzyme reaction was stopped and the color development was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Tokyo, Japan). The intensity of the color is inversely proportional to the concentration of PGE2 in either the standard dilutions or the samples.
Statistical analysis
Student's t-test was used to assess statistical significance of differences (Statview 5.0, SAS Institute Inc., Cary, USA). If p < 0.05, the difference was considered statistically significant.
Results
Effect of CA on proliferation of ovarian carcinoma cells
Our preliminary experiment confirmed that IC50 value of CDDP to OVCAR-3 cells was 0.5 μM/ml (Yokoyama et al. 1998). While addition of 50 μM CA alone in culture medium did not affect proliferation of the carcinoma cells, proliferation of the cells treated with 0.5 μM/ml CDDP alone or simultaneously treated with 0.5 μM/ml CDDP and 50 μM CA was significantly suppressed compared with the control and CA alone groups (p < 0.0001, respectively, Fig. 1). Furthermore, proliferation of the cells treated simultaneously with 0.5 μM/ml CDDP and 50 μM CA was significantly suppressed compared with that treated with 0.5 μM/ml CDDP alone (p < 0.05, Fig. 1).
Effect of CA on proliferation of the ovarian carcinoma cells. OVCAR-3 cells were incubated with 0.5 μM/ml CDDP in the presence or absence of 50 μM CA or incubated with 50 μM CA alone for 72 hr. Results were expressed as the percentage of untreated control (mean ± S.D., n = 3) from a single representative of three. * p < 0.0001 compared with the control and CA alone. ** p < 0.05 compared with CDDP alone.
Effect of CA on apoptosis induction in ovarian carcinoma cells
Chemotherapeutic effect of CDDP is based on its ability to induce apoptosis in tumor cells (Ormerod et al. 1994). To determine whether CDDP induces more apoptotic cells in the presence of CA than in the absence of CA, apoptotic cell death in different treatment was analyzed by Hoechst 33258 staining (Fig. 2a). Although treatment with CA alone did not affect the increase or decrease of the number of apoptotic cells, treatment with CDDP in the presence of CA significantly increased the number of apoptotic cells compared with that with CDDP alone (Fig. 2b).
Effect of CA on apoptosis induction by CDDP in OVCAR-3. Cells were cultured at 5 × 10
5
cells per 10 cm-diameter dish in growth medium containing 50 μM CA alone, 0.5 μM/ml CDDP alone or 0.5 μM/ml CDDP + 50 μM CA for 72 hr.
CA reduces PGE2 levels
Addition of CA in the culture medium significantly reduced PGE2 level compared to that in the absence of CA (Table 1). The concentration of PGE2 in the culture medium of the cells treated simultaneously with CDDP and CA was significantly lower than that in the culture medium of the cells treated with CDDP alone (Table 1).
CA reduces PGE2 level.
Results of PGE2 concentration are expressed as the mean value (mean ± S.D., n=3) from three separate experiments.
p < 0.05 compared to the control and treatment with CDDP alone.
Discussion
The present results showed that although 50 μM CA alone did not affect proliferation of the carcinoma cells, simultaneous treatment with 0.5 μM/ml CDDP and 50 μM CA significantly suppressed proliferation of the cells compared with that with 0.5 μM/ml CDDP alone. It also emerged that treatment with CDDP in the presence of CA significantly increased the number of apoptotic 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. The recent report showed 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 (Barnes et al. 2007). We recently demonstrated that addition of CA in the ovarian carcinoma cells caused a dose-dependent enhancement of CR expression (Yokoyama et al. 2007). CR is a cytoplasmic enzyme expressed in the lung, liver, kidney, and ovary, and its main function is to reduce carbonyl compounds with NADPH (Wermuth, 1981). CR specially promotes conversion of PGE2 to PGF2α (Schieber et al. 1992). Taken together, the present results suggest that CA could decrease PGE2 level through induction of CR, resulting in enhancement of antiproliferative effect of CDDP.
PGs play an important role in modulating tumor growth and metastasis in a variety of human tumors (Ablin and Shaw, 1986). In particular, PGE2 has been shown to have important functions in inhibiting apoptosis (Sheng et al. 1998) as well as in inducing angiogenesis (Tsujii et al. 1998) in carcinoma cells. Sheng et al. (1998) reported that while treatment of human colon carcinoma cells with a highly selective cyclooxygenase (COX)-2 inhibitor decreased colony formation, this growth inhibition was reversed by treatment with PGE2. Additionally, they described that PGE2 inhibited apoptosis caused by COX-2 inhibitor through induction of Bcl-2 expression (Sheng et al. 1998). Taking these results together including ours, it is suggested that PGE2 level surrounding the carcinoma cells affects tumor growth or modulation of apoptosis. Recently, the possibility has been reported that concentration of PGE2 might affect the sensitivity of the carcinoma cells to anticancer agents as well as cell proliferation and apoptosis induction (Hashitani et al. 2003). CDDP is known to cause a significant reduction in the mRNA levels and activities of peroxisomal enzymes (Portilla et al. 2002). PPARα ligands induce the activity and expression of peroxisomal enzymes. Li et al. (2004) reported that WY14643, PPARα ligand, prevented CDDP-induced reduction of mRNA levels and enzyme activity of peroxisome enzymes. Overall, these reports provide the evidence that the presence of CA in treatment with CDDP acts as a sensitizer of the carcinoma cells to the anticancer agent through reduction of PGE2 level. One study has suggested that CA action might be associated with intracellular reactive oxygen species (ROS) formation which could contribute to cell death (Qu et al. 2001). Increased apoptosis by treatment with CDDP in the presence of CA might be involved in increased ROS formation and oxidative stress.
The present study clarified the ability of CA to enhance a suppressive effect of CDDP on proliferation of the ovarian carcinoma cells. CA induced CR expression in the ovarian carcinoma cells and consequently increased an antiproliferative effect of CDDP via reduction of PGE2 concentration. CA would be promising as an enhancer of antitumor effect in combination with chemotherapeutic agents such as CDDP, although in vivo additional studies are needed to further define the mechanisms of the enhanced antitumor effect of CDDP by CA.
Footnotes
Acknowledgement
This study was supported in part by a Grant-in Aid for Cancer Research (No. 16591632) from the Ministry of Education, Science and Culture of Japan and by the Karoji Memorial Fund of the Hirosaki University School of Medicine.