Endometrial cancer is the fourth largest malignancy of women in the United States, and approximately 46,470 new cases were estimated to be diagnosed in 2011.1 While early-stage endometrial cancer (EC) has a favorable prognosis, advanced or recurrent EC presents a major challenge despite multimodal treatment.
Progesterone and its synthetic form, medroxyprogesterone acetate (MPA), have been recognized as an effective treatment option for patients with advanced and recurrent EC and those who wish to preserve the potential for fertility.2,3 The cellular responses of progestin are achieved by its binding to human progesterone receptor (PR) including PR-A and PR-B. Unfortunately, the down-regulation of PR isoforms is a frequent occurrence in endometrial carcinogenesis and is a principal cause of the failure of progestin for EC treatment.4,5 Moreover, progestin reduces the expression of progesterone receptor, and current long-term progesterone treatment strategies may lead to the resistance of EC to hormone therapy.6 Therefore, elevating or restoring PR expression in patients with EC presents an attractive approach to improve the efficacy of current therapeutic regimens.
Recently, epigenetic modifications, specifically DNA methylation, have been shown to be crucially involved in carcinogenesis. Aberrant hypermethylation of promoter CpG islands of the genes encoding tumor suppressors and steroid receptors has been detected in EC.7 Notably, DNA hypermethylation plays an important role in the down-regulation of PR in EC.8,9 5-Aza-2′-deoxycytidine (5-aza-CdR), a DNA methyltransferase inhibitor, has been approved by the US Food and Drug Administration for the treatment of myelodysplastic syndromes and is currently under evaluation in phase 1 and 2 clinical trials for the treatment of solid tumors.10,11 Furthermore, accumulating evidence indicated that 5-aza-CdR increased the antitumor efficacy of some chemotherapeutic agents against several cancers.12,13
Based on these previous studies, we investigated whether 5-aza-CdR is capable of sensitizing EC cells to MPA in vitro and evaluated the antitumor effect of the new treatment strategy that combines 5-aza-CdR with MPA.
MATERIALS AND METHODS
Cell Culture and Drug Treatment
The human EC cell line KLE (PR negative) was purchased from American Type Culture Collection (ATCC, VA), and Ishikawa cells (PR positive) were kindly provided by Dr Wei LH (Department of Gynecology and Obstetrics, Peking University People’s Hospital, China). Both cell lines were maintained in phenol red-free Dulbecco modified Eagle medium/F12 medium (Sigma-Aldrich, MO) supplemented with 10% dextran-coated charcoal–stripped fetal bovine serum and 0.1% antibiotics (penicillin G/streptomycin). The cells were cultured at 37°C in a humidified atmosphere of 5% CO2. Medroxyprogesterone acetate, 5-aza-CdR, and RU486 were purchased from Sigma-Aldrich. At approximately 30% confluence, the cells were treated by vehicle (dimethyl sulfoxide); MPA alone (10−8, 10−7, 10−6, and 10−5 mol/L) for 72 hours; 5-aza-CdR alone (0.1, 0.5, 1.5, and 5 μmol/L) for 72 hours; MPA for 72 hours after pretreatment with 5-aza-CdR for 72 hours. Culture media containing different drugs were refreshed every 24 hours.
Methylation-Specific Polymerase Chain Reaction (MSP)
The regions of PR-A and PR-B (AUG1 at +160 to +258 and AUG2 at −595 to −396 base pairs), which were close to their promoters, were analyzed. Genomic DNA was isolated using Wizard Genomic DNA Isolation Kit (Promega, WI). Two micrograms of DNA was subjected to sodium bisulfate conversion using an EZ DNA Methylation Kit (ZYMO Research, USA). Polymerase chain reaction was performed using the primers described previously10; 4 μL of converted DNA were eluted from DNA affinity column and mixed with 2 μL of deoxyribonucleotide triphosphate mix, 2.5 μL of AmpliTaq Gold 360 buffer ×10, 1 μL of magnesium chloride (25 mmol/L), 4 μL of 360 guanine cytosine enhancer, and 0.125 μL of AmpliTaq Gold 360 DNA polymerase (Applied Biosystems Inc, CA) in a total volume of 25 μL. Polymerase chain reaction products were resolved in 2% agarose gels and visualized by ethidium bromide staining.
Real-Time Fluorescence Quantitative PCR
Total RNA was extracted from the cells using Trizol reagent (Invitrogen, CA).
Complementary DNA was synthesized with 2 μg of RNA using Moloney murine leukemia virus reverse transcriptase, Oligo(dT)15 primer and deoxyribonucleotide triphosphate mix (Promega). To avoid genomic DNA contamination, RNA samples were incubated with ribonuclease-free deoxyribonuclease I (Promega) for 30 minutes at 37°C. As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were amplified from the same complementary DNA samples. Polymerase chain reaction was performed using real-time SYBR-green premix kit (TaKaRa, Liaoning, China) in combination with a fluorescence-based real-time detection method (ABI Prism 7500 real-time polymerase chain reaction (PCR) system, Taqman; Applied Biosystems, CA). The sequences of the primers were shown in supplemental data, (Supplemental Digital Content 1, http://links.lww.com/IGC/A93). The messenger RNA (mRNA) levels of progesterone receptor (PR), PR-B, and GAPDH were detected simultaneously for the same experimental sample using the respective standard curve. Because PCR results for total PR and PR-B were quantitated for each experimental sample, the amount of PR-A was determined by subtracting the value for PR-B from total PR.
Western Blot Analysis
The cells were lysed in lysis buffer, and cell extracts were prepared as described previously.14 Twenty micrograms of protein extracts were separated on each lane of sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to the membranes. The membranes were probed with PR or GAPDH primary antibody (CST, MA) and goat antirabbit horseradish peroxidase–linked secondary antibody (CST). Bands were visualized by enhanced chemiluminescence reagents (Pierce, IL). The relative protein levels were normalized to GAPDH and quantified by densitometry with AlphaEasyFC software (Alpha Innotech, CA) and expressed as the ratio to the untreated control subjects.
Cell Proliferation Assay
The proliferation of EC cells was measured by 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) cell proliferation kit (Roche Diagnostics, Mannheim, Germany). Exponentially growing cells were harvested and seeded in 96-well plates (Nunclon, NY) for 24 hours. After a 24-hour serum starvation, cells were exposed to the chemicals and WST-1 assay was performed according to the manufacturer’s instructions. Cell growth was determined using the microplate reader (Bio-Tek, USA) at 450/690-nm wavelength. Absorbance values of experimental cultures were used to indicate the levels of cell proliferation. The percentage of cytotoxicity was calculated using the following formula:
Each experiment was performed in duplicate and repeated 3 times independently.
Cell Cycle Analysis
The cells (2 × 105) were plated in 60-mm tissue culture plates and cultured overnight, then the cells were serum starved overnight for synchronization. Next, the cells were treated by the agents as indicated and harvested for flow cytometry analysis. Briefly, the cells were fixed with 300-μL phosphate-buffered saline and 700-μL ethanol at 4°C overnight. Then the cells were stained with DNA staining solution containing 50-μg/mL propidium iodide and 1-mg/mL RNase-A at 37°C for 30 minutes in the dark. Finally, the cells were analyzed with a FACSCalibur flow cytometer (Becton, Dickinson and Co, CA), and the data were analyzed with ModfitLT 3.0 software (Verity Software House, ME).
Cell invasion assay was performed with 24-well transwell chamber (8 μm; Costa, Lowell, MA) containing 20-μg matrigel (BD Biosciences, NJ). Briefly, the suspensions of the cells treated with the agents (3 × 104) were plated in the upper chamber, and 500 μL of 1% dextran-coated charcoal–stripped fetal bovine serum in Dulbecco modified Eagle medium/F12 was added in the lower chamber. The cells were incubated at 37°C in 5% CO2 for 24 hours, and the noninvading cells were removed from the upper chamber with a cotton swab. The invading cells in the lower chamber were fixed in 0.25% glutaraldehyde and stained with hematoxylin-eosin. After washing with phosphate-buffered saline, the stained cells were counted under microscope (Olympus IX51 at ×200) in 5 different fields. The assay was performed 3 times independently.
Latent and active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium were detected by zymography on 10% sodium dodecyl sulfate–polyacrylamide gels containing 1-mg/mL denatured collagen (gelatin, G2500, Sigma) under nonreducing conditions as described previously.15 Gels were stained with Coomassie brilliant blue for 20 minutes, and destained for 1 hour in 30% methanol/10% acetic acid (vol/vol). The proteolytic activity was visualized by negative staining. Relative activities of band intensity were quantified with densitometric analysis of zymograms using AlphaEasyFC software (Alpha Innotech, CA).
Median Effect Analysis
For median effect analysis the combination index (CI) was calculated by
to examine the synergism and the additivity/antagonism between 5-aza-CdR and MPA. Fa-CI Plot (Chou-Talalay Plot) was used for visual illustration. Combination indexes less than 1, 1, and greater than 1 indicated synergism, additive effect, and antagonism, respectively.
All statistical analyses were performed with SPSS 13.0 software. Differences between the treatment groups were analyzed by one-way analysis of variance. P < 0.05 was considered statistically significant.
5-Aza-CdR Synergizes With MPA to Inhibit the Growth of EC Cells
WST-1 assay showed that the proliferation of Ishikawa and KLE EC cells decreased in a dose-dependent manner after treatment with 5-aza-CdR. The half-maximal inhibitory concentration (IC50) of 5-aza-CdR was 3.19 μmol/L in Ishikawa cells and 7.91 μmol/L in KLE cells. Meanwhile, Ishikawa cells showed a good response to MPA, whereas KLE cells were resistant to MPA (Figs. 1A, B). Furthermore, we demonstrated that 5-aza-CdR significantly enhanced the susceptibility of EC cells to MPA. Medroxyprogesterone acetate alone at the concentration of 10−6 mol/L showed 6.7% growth inhibition in KLE cells, but MPA together with 0.5-μmol/L and 1.5-μmol/L 5-aza-CdR led to a growth inhibition of 38.2% and 59.2% in KLE cells, respectively. In Ishikawa cells, 10−6-mol/L MPA led to 46.8% growth inhibition, but MPA together with 0.5-μmol/L and 1.5-μmol/L 5-aza-CdR resulted in growth inhibition of 51.8% and 68.1%, respectively (Figs. 1A, B). To determine the interaction between 5-aza-CdR and MPA, the CI for the drugs at different concentrations was calculated using CalcuSyn software. The results showed that the 2 drugs synergized to reduce the proliferation of both EC cell lines, and the synergistic effect was the strongest when 0.1-μmol/L 5-aza-CdR was combined with 10−7-mol/L MPA in Ishikawa cells or 5.0-μmol/L 5-aza-CdR was combined with 10−6-mol/L MPA in KLE cells (CIs were 0.30781 and 0.0263 in Ishikawa and KLE cells, respectively; Table 1).
5-Aza-CdR Synergizes With MPA to Induce Cell Cycle Arrest and Apoptosis of EC Cells
Because 5-Aza-CdR synthesizes with MPA to inhibit the growth of EC cells, next we examined cell cycle and apoptosis of EC cells treated by these 2 agents. Flow cytometry analysis showed that 5-Aza-CdR induced a significant G2/M block and increased the subdiploid population (indicating apoptosis) in Ishikawa cells, which was less obvious in the KLE cells (Figs. 2A, B). Medroxyprogesterone acetate at a concentration of 10−6 mol/L had little impact on the cell cycle of both cell lines (Figs. 2A, B). However, combination treatment led to increased percentages of cells at G2/M arrest (Fig. 2C) and increased percentages of subdiploid population in both cell lines (Fig. 2D). These results suggest that 5-Aza-CdR synergizes with MPA to inhibit the growth of EC cells via the induction of cell cycle arrest and apoptosis.
5-Aza-CdR Induces Demethylation of PR-B Promoter and Expression of PR in EC Cells
We analyzed the induction of PR expression by 5-aza-CdR in Ishikawa and KLE cell lines. Methylation-specific PCR revealed that the PR-B promoter was methylated, where as the PR-A promoter was unmethylated in these cells. Only partial demethylation of the PR-B promoter was detected after 0.1-μmol/L 5-aza-CdR treatment over 3 days, but high concentration (0.5 and 1.5 μmol/L) treatment induced a further decrease in the methylation of the PR-B promoter (Figs. 3A, B).
Consistent with our observation that 5-Aza-CdR induced the demethylation of the PR-B promoter, real-time PCR and Western blotting analysis showed that 5-Aza-CdR increased the expression of PR-B at both mRNA and protein levels in both cell lines in a dose-dependent manner (Figs. 3D, F). Surprisingly, 5-aza-CdR also increased the expression of PR-A in a dose-dependent manner, although the methylation of the PR-A promoter was not detected in both cell lines (Figs. 3C, E).
To further explore the effect of 5-aza-CdR on PR activity, the mRNA expression of progesterone-induced genes glycodelin and forkhead box protein O1 (FOXO1) was evaluated by real-time PCR. Medroxyprogesterone acetate at the concentration of 10−6 mol/L increased the mRNA expression of glycodelin and FOXO1 in Ishikawa cells but not in KLE cells. However, 5-aza-CdR plus MPA induced the up-regulation of glycodelin and FOXO1 expression in both Ishikawa and KLE cells (Figs. 3G, H).
5-Aza-CdR Synergizes With MPA to Inhibit Invasion of EC Cells
To investigate the synergistic effects of 5-aza-CdR and MPA on the invasion behaviors of EC cells, we performed in vitro invasion assay. The results showed that MPA (10−6 mol/L) significantly inhibited the invasive potential of Ishikawa cells but had only a slight effect on the invasion of KLE cells. Notably, the addition of 5-aza-CdR significantly enhanced the inhibition of KLE cell invasion by MPA, but 5-aza-CdR and MPA did not produce a further inhibitory effect on Ishikawa cell invasion (Fig. 4).
Elevated expression and activation of MMP-2 and MMP-9 are closely related to the aggressiveness and progression of endometrial cancers.16 Thus, we measured the expression and gelatinolytic activity of MMP-2 and MMP-9 in Ishikawa and KLE cells. Real-time PCR analysis demonstrated that the combination of 5-aza-CdR and MPA obviously decreased the mRNA expression of MMP-2 and MMP-9 in both EC cells. In addition, combination treatment significantly suppressed the mRNA expression of MMP-2 in KLE cells compared with MPA treatment alone (Figs. 5A, B). Gelatin zymography analysis further showed that the combination of MPA and 5-aza-CdR reduced the activities of MMP-2 and MMP-9 in Ishikawa and KLE cells (Figs. 5C, D).
In the present study, we present the novel finding that 5-aza-CdR could enhance progestin sensitivity in EC cells, and it showed synergistic antiproliferation and anti-invasion effects on EC cells when combined with MPA.
5-Aza-2′-deoxycytidine is known to inhibit the growth of several solid cancer cells in vitro.17,18 Our data showed a dose-dependent inhibitory effect of 5-aza-CdR on EC cell growth, consistent with previous report.19 5-Aza-2′-deoxycytidine suppresses the proliferation of tumor cells by acting as a chemotherapeutic agent and causes DNA damage because of the structural instability at its incorporation sites.20 Perhaps owing to the different duplication time of Ishikawa cells (35.3 hours) and KLE cells (109.9 hours), their proliferation was influenced to different extent by 5-aza-CdR, which suggests that the growth-inhibitory action of 5-aza-CdR is closely related to the cell growth rate. Furthermore, the expression level of PR in KLE cells was much lower than in Ishikawa cells, indicating that the PR status could be another contributing factor.
Despite being a highly effective anticancer drug, the toxicity of 5-aza-CdR at higher doses can cause myelosuppression and intestinal mucosal necrosis and other adverse effects.16,21 Therefore, lower concentrations of 5-aza-CdR, which can be tolerated in patients with cancer, were used in this combination treatment study.22 Our results demonstrated that 5-aza-CdR significantly enhanced the susceptibility of EC cells to MPA. In KLE cells, 10−6-mol/L MPA showed little growth inhibition, whereas more than 50% growth inhibition was observed in combination with 5-aza-CdR. In contrast, Ishikawa cells had a good response to MPA, and the increased effect on growth inhibition by MPA and 5-aza-CdR was less obvious. Moreover, 5-aza-CdR and MPA exhibited a synergistic effect (CI <1) to inhibit the proliferation of both EC cell lines. Preincubation with 5-aza-CdR (0.5 μmol/L) significantly reduced the IC50 values of MPA in Ishikawa cells (from 10−6 to 10−7 mol/L) and KLE cells (from 4 × 10−3 to 10−6 mol/L). Taken together, these results indicate that pretreatment with low concentration of 5-aza-CdR is sufficient to increase the chemosensitivity of EC cells.
Cells are sensitized to progesterone through the PR pathway.23 We observed that MPA had significant inhibitory effects on the growth of Ishikawa but not KLE cells, which confirmed that PR-B is a major factor that affects the progestin-inhibitory effect on endometrial cancer. Our results showed that 5-aza-CdR induced a dose-dependent demethylation of PR-B, accompanied with increased expression of PR-B. These results, together with data from Xiong et al,24 suggest that the sensitizing effect of 5-aza-CdR in EC cells depends on the level of PR-B, but the role of PR-A remains unclear although the expression of PR-A was up-regulated by 5-aza-CdR as well, consistent with a previous study by Ren et al.25 Whereas we did not detect demethylation in the screened regions of PR-A after 5-aza-CdR treatment, other CpG sites that we did not examine may be involved. Furthermore, as a global DNA methyltransferase inhibitor, 5-aza-CdR could regulate indirectly the expression of genes such as estrogen receptor.26
Progesterone receptor is a nuclear transcription factor that regulates the transcription of target genes in a ligand-responsive manner. Previous studies have shown that PR isoforms exhibit different properties and mediate the transcription of a set of genes including glycodelin and FOXO1.27,28 We confirmed that MPA increased the mRNA expression of glycodelin and FOXO1 in EC cells. Interestingly, the mRNA levels of glycodelin and FOXO1 were much higher after combined treatment with 5-aza-CdR and MPA in both Ishikawa and KLE cells. This observation demonstrated that 5-aza-CdR enhanced the PR-mediated gene transcription.
Previous studies reported that 5-aza-CdR induced cell cycle arrest and apoptosis in several cancer cells.29,30 Our study demonstrated that the treatment of EC cells with 5-aza-CdR induced G2/M arrest and increased subdiploid population (apoptosis). In contrast, the cell cycle was not influenced by 10−6-mol/L MPA. In cells treated with the combination of 5-aza-CdR and MPA, a greater percentage of cells in the G2/M and sub-G1 phases were observed compared to cells treated with either drug alone. These findings provide further evidence of a synergistic action between 5-aza-CdR and MPA against EC cells.
Effective cancer therapeutics should inhibit not only primary tumor growth but also tumor metastasis to distant organs. Therefore, we further explored whether 5-aza-CdR had an effect on the invasion of EC cells. Our results demonstrated that 5-Aza-CdR synergizes with MPA to inhibit the invasion of EC cells, and the mechanism may be related to the inhibition of MMP-2 and MMP-9 expression and activity in EC cells as revealed by RT-PCR and gelatin zymography analysis.
In conclusion, we propose that low dose of 5-aza-CdR could enhance the antitumor efficacy of MPA in EC cells, particularly in the PR-negative EC cells. The beneficial effect of 5-aza-CdR is mediated by the up-regulation of PR expression and the inhibition of endometrial cancer cell proliferation and invasion. Our findings suggest that patients with progestin resistance and a rapid tumor growth rate might be more suitable to receive this combined treatment.
The authors are grateful to Dr Ting-Chao Chou (Memorial Sloan-Kettering Cancer Center, USA) for the CalcuSyn software.
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Keywords:Copyright © 2012 by IGCS and ESGO
Endometrial cancer; Progesterone receptor; 5-aza-2′-deoxycytidine; Medroxyprogesterone acetate; DNA methylation