15-Deoxy-Δ12,14-prostaglandin J2 induces growth inhibition, cell cycle arrest and apoptosis in human endometrial cancer cell lines
Affiliations: Department of Obstetrics and Gynecology, Oita University Faculty of Medicine, Yufu-shi, Oita, Japan
- Published online on: February 5, 2013 https://doi.org/10.3892/ijmm.2013.1268
- Pages: 778-788
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Endometrial cancer is the most common malignant tumor of the female genital tract, and its incidence has increased in recent years (1,2). Furthermore, the search for agents effective in the treatment of either advanced or recurrent endometrial cancer has proved to be disappointing (2,3). Therefore, innovative approaches are required for the treatment of endometrial cancer.
Peroxisome proliferator-activated receptor (PPAR)γ is a nuclear hormone receptor and its ligands, troglitazone and pioglitazone, have been shown to induce apoptosis in several types of cancer cells, including endometrial cancer cells (4–6). 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is a PPARγ ligand that activates PPARγ at micromolar concentrations in humans in vivo (7–9). Recently, 15d-PGJ2 was reported to have antiproliferative activity in certain types of cancer (4,10–12). However, the effect of 15d-PGJ2 on endometrial cancer cells has not yet been investigated.
The present study aimed to investigate the biological and therapeutic effects of 15d-PGJ2 on endometrial cancer. We examined whether this compound can mediate cell growth inhibition, cell cycle arrest and apoptosis in endometrial cancer cell lines (HHUA, Ishikawa and HEC-59). Furthermore, to identify potential and novel target genes responsive to the anticancer effect in 15d-PGJ2-treated endometrial cancer cells, we analyzed the global changes in gene expression in HHUA cells following treatment with 15d-PGJ2 using cDNA microarrays. The expression of candidate proteins was confirmed by western blot analysis in the 3 endometrial cancer cell lines.
Materials and methods
The HHUA human endometrial cancer cell line was obtained from Riken (Ibaraki, Japan). The Ishikawa human endometrial cancer cell line was kindly provided by Dr Masato Nishida (Tsukuba University, Ibaraki, Japan). The HEC-59 human endometrial cancer cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained as monolayers at 37°C in 5% CO2/air in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Rockville, MD, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Omega, Tarzana, CA, USA).
15d-PGJ2 was obtained from Enzo Life Sciences (Plymouth Meeting, Montgomery County, PA, USA), and prepared as a 20 mg/ml stock solution in dimethyl sulfoxide (DMSO). The stock solution was stored in aliquots at −20°C.
Assessment of cell proliferation and cell viability
The cell proliferation and cell viability were determined in 96-well plates by a modified methylthiazol tetrazolium (MTT) assay using WST-1 (Roche Diagnostics, Penzberg, Germany) following the manufacturer’s instructions. We distributed 5×103 cells in DMEM supplemented with 10% FBS into each well of a 96-well flat-bottomed microplate (Corning, Inc., New York, NY, USA) and incubated them overnight. The medium was then removed, and the cells were incubated for 48 h with 100 μl of experimental medium containing various concentrations of 15d-PGJ2. Thereafter, 10 μl of WST-1 dye was added to each well, and the cells were further incubated for 4 h. All experiments were performed in the presence of 10% FBS. Cell proliferation was evaluated by measuring the absorbance at 540 nm. Data were calculated as the ratio of the values obtained for the 15d-PGJ2-treated cells to those for the untreated controls.
Cell cycle analysis by flow cytometry
The cell cycle was analyzed by flow cytometry after 2 days of culturing. Cells (5×104) were exposed to 15d-PGJ2 in 6-well flat-bottomed plates for 48 h. Analysis was performed immediately after staining using the CellFIT program (Becton-Dickinson, San Jose, CA, USA), whereby the S phase was calculated using an RFit model.
Measurement of apoptosis [flow-cytometric analysis with the Annexin V/propidium iodide (PI) assay]
Cells were plated and grown overnight until they reached 80% confluence and then treated with 15d-PGJ2. After 48 h, detached cells in the medium were collected, and the remaining adherent cells were harvested by trypsinization. The cells (1×105) were washed with PBS and resuspended in 250 μl of binding buffer (Annexin V-FITC kit; Becton-Dickinson) containing 10 μl of 20 μg/ml PI and 5 μl of Annexin V-FITC, which binds to phosphatidylserine translocated to the exterior of the cell membrane early in the apoptotic pathway as well as during necrosis. After incubation for 10 min at room temperature in a light-protected area, the samples were analyzed on a FACSCalibur flow cytometer (Becton-Dickinson). FITC and PI emissions were detected in the FL-1 and FL-2 channels, respectively. For each sample, data from 30,000 cells were recorded in list mode on logarithmic scales. Subsequent analysis was performed with CellQuest software (Becton-Dickinson).
Mitochondrial transmembrane potential (MTP)
Cells were prepared for FACS analysis as described above and stained using a Mitocapture Apoptosis Detection kit obtained from BioVision (Palo Alto, CA, USA) with a fluorescent lipophilic cationic reagent that assesses mitochondrial membrane permeability, according to the manufacturer’s recommendations.
Total RNA was extracted from the 15d-PGJ2-treated and untreated HHUA cells using an RNeasy mini kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s instructions. Prior to hybridization, the quantity and quality of the total RNA were evaluated using a spectrophotometer and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. Cy3-labeled cRNA targets were generated using a Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies). A human 44 K oligoarray was used for hybridization, in accordance with the manufacturer’s recommendations (Agilent Technologies). A laser confocal scanner (Agilent Technologies) was used to measure signal intensities in the expression microarray analysis. Feature Extraction software (Version 9.1; Agilent Technologies) with the manufacturer’s recommended settings was applied for the microarray image analysis. Analysis of the microarray images was performed with GeneSpring 7.3.1 software (Agilent Technologies). For comparison among multiple arrays, probe set data were median-normalized/chip. The data were then centered across the genes in 6 normal controls, followed by filtering based on a signal intensity of ≥100, and contained no flagged values. Among these differentially expressed genes, those designated as ‘upregulated’ were overexpressed >2-fold in comparison with the controls (P<0.05), whereas those designated as ‘downregulated’ were underexpressed <0.75-fold compared with the controls (P<0.05). Annotations including chromosomal loci were provided by Agilent Technologies.
For Gene Ontology (GO) analysis, differentially expressed genes were defined as those with a >2-fold increase or decrease in expression relative to the controls. GO term enrichment in the upregulated or downregulated gene sets was assessed using the GOstat web tool (13).
Western blot analysis
Cells were washed twice in PBS, suspended in lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, phenylmethylsulfonyl fluoride at 100 μg/ml, aprotinin at 2 μg/ml, pepstatin at 1 μg/ml and leupeptin at 10 μg/ml], and placed on ice for 30 min. After centrifugation at 15,000 × g for 15 min at 4°C, the suspension was collected. Protein concentrations were quantified using the Bio-Rad protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s recommendations. Whole-cell lysates (40 μg) were resolved by SDS-polyacrylamide gel electrophoresis on a 4–15% gel, transferred onto a polyvinylidene difluoride membrane (Immobilon; Amersham, Arlington Heights, IL, USA), and probed sequentially with antibodies against anterior gradient homolog 3 (AGR3; 1:1,000; GeneTex, Irvine, CA, USA), aldo-keto reductase family 1 member C1 (AKR1C1; 1:1,000; GeneTex), aldo-keto reductase family 1 member C3 (AKR1C3; 1:1,000; ProteinTech, Chicago, IL, USA), α-1-microglobulin/bikunin precursor (AMBP; 1:1,000; Abnova, Taipei, Taiwan), complement component 3a receptor 1 (C3AR1; 1:1,000; Abnova), chondroadherin (CHAD; 1:1,000; Avia Systems Biology, San Diego, CA, USA), Fer3-like (Drosophila) (FERDL3; 1:1,000; Avia Systems Biology), ferritin, light polypeptide (FTL; 1:1,000; GeneTex), galactose-3-O-sulfotransferase 3 (GAL3ST3; 1:1,000; Avia Systems Biology), glutamate-cysteine ligase, modifier subunit (GCLM; 1:1,000; Abnova), heme oxygenase (decycling) 1 (HMOX1; 1:1,000; Abnova), intercellular adhesion molecule 4 (ICAM4; 1:1,000; Abnova), potassium voltage-gated channel, shaker-related subfamily, β member 1 (KCNAB1; 1:1,000; Osenses, Keswick, Australia), mitochondrial ribosomal protein L37 (MRPL37; 1:1,000; ProteinTech), nitric oxide synthase 2A (NOS2A; 1:1,000; Applied Biological Materials, Kampenhout, Belgium), phosphorylated eukaryotic translation initiation factor 4E (p-eIF4E; 1:1,000; Bioworld Technology, Minneapolis, MN, USA), pirin (PIR; 1:1,000; Avia Systems Biology), tripartite motif-containing 16 (TRIM16; 1:1,000; Avia Systems Biology), thioredoxin reductase 1 (TXNRD1; 1:1,000; ProteinTech), UDP glucuronosyltransferase 1 family, polypeptide A6 (UGT1A6; 1:1,000; LifeSpan Biosciences, Seattle, WA, USA) and GAPDH monoclonal antibody (mAb) (1:10,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The blots were developed using an enhanced chemiluminescent (ECL) kit (Amersham). Band intensity was measured using the public domain Image program ImageJ version 1.44, and fold increase in expression as compared with control, untreated cells was calculated.
Data are presented as the means ± SD of representative experiments and were analyzed by the Bonferroni-Dunn test using StatView 4.5 software (Abacus Concepts, Berkeley, CA, USA). A P-value <0.05 was considered to indicate a statistically significant difference.
Effects of 15d-PGJ2 on the proliferation and viability of endometrial cancer cell lines in vitro
The antitumor effects of 15d-PGJ2 on 3 endometrial cancer cell lines in vitro were examined using a WST-1 assay of the 2-day exposure to 15d-PGJ2. Significant inhibitory effects of 15d-PGJ2 on the cell growth were observed in all 3 endometrial cancer cell lines (Ishikawa, HHUA and HEC-59) (Fig. 1).
Effect of 15d-PGJ2 on the growth of endometrial cancer cells in vitro. Ishikawa, HEC-59 and HHUA endometrial cancer cells were treated with either 15d-PGJ2 at various concentrations (5–20 μM) or the dilutant (control) for 48 h, and cell growth (% of control) was measured using an WST-1 assay. Results represent the means ± SD of 3 independent experiments with triplicate dishes.
Cell cycle analysis of endometrial cancer cells following exposure to 15d-PGJ2
We then investigated whether 15d-PGJ2 would lead to the induction of apoptosis and/or cell cycle arrest in the endometrial cancer cells (Table I). 15d-PGJ2 led to an increase in the sub G0/G1 apoptotic cell population and the cell population in the G2/M phase of the cell cycle compared to treatment with the vehicle alone, with a concomitant decrease in the proportion of cells in the S phase.
Apoptotic changes in endometrial cancer cells treated with 15d-PGJ2
To assess the ability of the endometrial cancer cells to undergo apoptosis in response to 15d-PGJ2 exposure and to distinguish between the different types of cell death, we double-stained the 15d-PGJ2-treated cells with Annexin V and PI and analyzed the results using flow cytometry. Annexin V binding combined with PI labeling was performed for the distinction of early apoptotic (Annexin V+/PI−) and necrotic (Annexin V+/PI+) cells. At increasing doses of 15d-PGJ2, a simultaneous increase in both the Annexin V+/PI− fraction (early apoptotic) and Annexin V+/PI+ (regarded as necrotic) subpopulations was detected (Table II).
Cell death measured by Annexin V and mitochondrial transmembrane potential assay in endometrial cancer cell lines.
Loss of MTP in response to treatment with 15d-PGJ2
It has been shown that the loss of MTP occurs prior to nuclear condensation and caspase activation and is linked to cytochrome c release in many, but not all, apoptotic cells (14,15). It was found that the treatment of endometrial cancer cells with 15d-PGJ2 resulted in the loss of MTP (Table II).
Differential gene expression in 15d-PGJ2-treated cells
In order to identify potential and novel target genes responsive to the anticancer effects in 15d-PGJ2-treated endometrial cancer cells, we examined the global changes in gene expression in the HHUA cells following treatment with 10 μM of 15d-PGJ2 for 48 h (Tables III and IV). Of the 44,000 genes, GO analysis was carried out on the genes upregulated and downregulated by the treatment (Tables V and VI).
Permutation analysis of the correlation between GO terms and upregulated genes following treatment with 15d-PGJ2.
Permutation analysis of the correlation between GO terms and downregulated genes following treatment with 15d-PGJ2.
Effects of 15d-PGJ2 on the expression of novel proteins
To elucidate the common mechanism of action of 15d-PGJ2 in endometrial cancer, we examined the effects of 15d-PGJ2 on the expression of 20 proteins that were selected from the cDNA microarray data in 3 endometrial cancer cell lines using western blot analysis (Fig. 2 and Table VII). 15d-PGJ2 markedly upregulated the levels of AKR1C3 and downregulated the levels of AGR3 and NOS2A proteins in all 3 endometrial cancer cell lines.
Expression of 20 proteins in endometrial cancer cells measured by western blot analysis. Cells were treated with 10 μM 15d-PGJ2, and cell lysates were harvested after 48 h. Western blot analysis was performed with a series of antibodies. The control cells were treated with the vehicle alone. The amount of protein was normalized by comparison to GAPDH levels. Lane 1, HHUA controls; lane 2, HHUA cells treated with 15d-PGJ2; lane 3, Ishikawa controls; lane 4, Ishikawa cells treated with 15d-PGJ2; lane 5, HEC-59 controls; lane 6, HEC-59 cells treated with 15d-PGJ2.
In the present study, we demonstrated that 15d-PGJ2 inhibits cell viability in endometrial cancer cells. The prominent arrest of these cells in the G2/M phase of the cell cycle and the induction of apoptosis likely account for this inhibitory effect, suggesting that 15d-PGJ2 has anticancer activity.
In order to investigate the molecular mechanisms involved in the effects of 15d-PGJ2 on the cell cycle arrest and the induction of apoptosis, we investigated the global gene expression profile changes in HHUA endometrial cancer cells following treatment with 15d-PGJ2. Surprisingly, the expression of PPARγ or angiotensin II type 1 receptor (AT1R) was not altered, although 15d-PGJ2 has been characterized as a potent PPARγ ligand. To identify novel target genes of 15d-PGJ2, we focused on some GO terms of the numerous genes upregulated and downregulated by 15d-PGJ2 treatment in the HHUA cells. GO analysis revealed that oxidation reduction (GO:0055114) and oxidoreductase activity (GO:0016491) were enriched in genes that were overexpressed in the 15d-PGJ2-treated HHUA cells compared to the untreated HHUA cells. Both GO terms include AKR1C3.
AKR1C3 is a multifunctional enzyme involved in androgen, estrogen, progesterone and prostaglandin metabolism. AKR1C3-mediated steroid metabolism may play a critical role in the maintenance of viable normal and abnormal endometrial epithelium (16). AKR1C3 has been reported to play important roles in the physiology of endometrial cells and that suppressed AKR1C3 expression represents a feature that allows the differentiation of hyperplastic and neoplastic endometrial epithelium from normal endometrial epithelium (16). In the present study, we demonstrated that 15d-PGJ2 markedly upregulated the levels of the AKR1C3 protein in all 3 endometrial cancer cell lines. Based on these observations, it can be hypothesized that the 15d-PGJ2-induced anticancer activity may be mediated, at least in part, by the upregulation of AKR1C3 in human endometrial cancer cells.
We confirmed the downregulation of AGR3 using western blot analysis in all 3 cell lines examined. AGR genes, a protein disulfide isomerase (PDI) family, harbour core thioredoxin folds (CxxS motifs) that have the potential to regulate protein folding and maturation. AGR3 is overexpressed by a hormone (estrogen-receptor α)-independent mechanism, identifying a novel protein-folding associated pathway that can mediate resistance to DNA-damaging agents in human cancers (17). These findings indicate that the downregulation of AGR3 by 15d-PGJ2 may cause DNA-damage, leading to the apoptosis of endometrial cancer cells.
Nitric oxide, a reactive free radical, acts as a biological mediator in several processes, including neurotransmission and antimicrobial and antitumor activities. The NOS2A gene encodes a nitric oxide synthase which is expressed in the liver and is inducible by a combination of lipopolysaccharide and certain cytokines. A recent study revealed that NOS2 upregulation contributes primarily to the proliferation and tumor maintenance in highly tumorigenic human glioma stem cells (18). Therefore, our finding that 15d-PGJ2 downregulated NOS2A expression suggests that the eicosanoid may inhibit the proliferation and maintenance of endometrial cancer cells via NOS2A downregulation.
In conclusion, the data from the present study demonstrate that 15d-PGJ2 exhibits anti-proliferative activity, potently induces cell cycle arrest, and stimulates apoptosis in human endometrial cancer cells. These events were accompanied by the upregulation of AKR1C3 and the downregulation of AGR3 and NOS2A. It is suggested that 15d-PGJ2 may be a novel therapeutic option for the treatment of endometrial cancer.
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