A new trick for an old dog: The application of mifepristone in the treatment of adenomyosis

Abstract Adenomyosis is also called internal endometriosis and affects about 20% of reproductive‐aged women. It seriously reduces life quality of patients because current drug therapies face with numerous challenges. Long‐term clinical application of mifepristone exhibits wonderful therapeutic effects with mild side‐effects in many disorders since 1982. Since adenomyosis is a refractory disease, we investigate whether mifepristone can be applied in the treatment of adenomyosis. In this study, we investigated the direct effects of mifepristone on human primary eutopic endometrial epithelial cells and stromal cells in adenomyosis. We found that mifepristone causes cell cycle arrest through inhibiting CDK1 and CDK2 expressions and induces cell apoptosis via the mitochondria‐dependent signalling pathway in endometrial epithelial cells and stromal cells of adenomyosis. Furthermore, mifepristone inhibits the migration of endometrial epithelial cells and stromal cells through decreasing CXCR4 expression and restricts the invasion of endometrial epithelial cells via suppression of epithelial‐mesenchymal transition in adenomyosis. We also found that mifepristone treatment decreases the uterine volume, CA125 concentration and increases the haemoglobin concentration in serum for adenomyosis patients. Therefore, we demonstrate that mifepristone could serve as a novel therapeutic drug in the treatment of adenomyosis, and therefore, the old dog can do a new trick.

women's quality of life and leads to a heavy burden on the cost of health care. [3][4][5][6][7] The treatment options for adenomyosis are summarized by Soave et al 8 and mainly include medical treatment, high intensity focused ultrasound (HIFU), surgery and combination therapy. 8 Since hysterectomy is not accepted by most patients, drug therapy is still the firstline treatment for adenomyosis. 9 However, numerous challenges are emerging including notable side-effects, limited efficacy and heavy financial burden. Therefore, the novel treatments are still deserved to be investigated. Mifepristone is the first and one of the most widely used selective progesterone receptor modulator since 1982. 10 Until now, mifepristone has been proved to be effective in the treatment of endometriosis, uterine fibroids 11,12 and a variety of cancers including glioblastoma multiforme, lung cancer, cervical cancer, ovarian cancer and endometrial cancer. [13][14][15][16] Mifepristone exhibits wonderful clinical efficacies in more and more human diseases than what we originally thought following current understanding of mechanism of action of mifepristone. Long-term clinical application of mifepristone proves that it is well tolerated and its side-effects are generally mild.
Besides, mifepristone has a very low price of long-term treatment. 17 Since this old dog, mifepristone, exhibits so many advantages and adenomyosis is a refractory disease, we want to know whether it can have a new trick in the treatment of adenomyosis.
Adenomyosis results from the increased invasive characteristics of the eutopic endometrial cells including increased proliferation, decreased apoptosis and angiogenesis, and these changes enhance the migratory capacity of eutopic endometrial cells into the myometrium and lead to the excessive proliferation of ectopic endometrial cells in the myometrium. 18,19 So eutopic endometrial cells, including endometrial epithelial cells and stromal cells, play crucial roles in the initiation and progression of adenomyosis. 20 Obviously, drug therapy for adenomyosis should be based on the above-mentioned pathogenesis of adenomyosis. In this study, we investigated whether the old and wonderful drug could be applied for the treatment of this painful and refractory disease. Our study is performed to provide the laboratory evidence and clinical basis for the treatment of adenomyosis with mifepristone.

| Isolation and identification of endometrial epithelial and stromal cells of adenomyosis
Endometrial tissues were obtained from hysterectomy samples of adenomyosis patients. The collected tissue was put into a culture dish and washed with FBS-free medium. Tissues were minced into 1 × 1 × 1 mm 3 pieces and digested with 1 mg/mL collagenase type III 37°C for 60 minutes, and then, the endometrial cells were separated by two sequential filtrations of 200-and 70-μm cell strainer.
Subsequently, endometrial epithelial cells were re-suspended in primary epithelial growth medium (PriCells) and endometrial stromal cells were cultivated in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Thermo Fisher) supplemented with 10% FBS (Sigma-Aldrich). The isolated endometrial epithelial cells and stromal cells were identified by immunofluorescent staining using epithelial marker pancytokeratin and mesenchymal marker vimentin as described in our previous study. 21

| The cell-counting kit-8 assay
The viability of endometrial epithelial and stromal cells with mifepristone treatment was detected by the cell-counting kit-8 (CCK-8) assay (Biosharp). According to the manufacturer's protocol, the CCK-8 reagent was added to each well and cells were incubated at 37°C for 2 hours. The absorbance at 450 nm (optical density) was measured and used to represent the cell viability. Each experiment was performed in three parallel wells and repeated for three times.

| Cell migration assay
Cell migration ability was evaluated by transwell chamber assay using 24-well plates with 8.0-μm pore size membranes (BD Biosciences). To study the effect of mifepristone on the migratory ability of endometrial cells, endometrial epithelial and stromal cells were pre-treated with mifepristone or AMD3100 (the CXCR4 inhibitor) in different concentrations (0, 50, 100 and 200 μmol/L for mifepristone and 0, 0.1, 1.0 and 10 μg/mL for AMD3100, respectively) in this experiment. The same number of endometrial cells were added into the upper chamber of the insert in 200 μL of serum-free medium, while the lower chamber contained growth media with 10% FBS. After 24-hour incubation, cells in the upper chamber were removed with a cotton swab and the migrated cells in the lower chamber were fixed with methanol, stained with crystal violet and counted with a microscope (Olympus). The number of cells that passed through the membrane was defined as migrated cell number.

| High-throughput sequencing
Primary endometrial epithelial cells were treated with mifepristone in three different concentrations (0, 50 and 100 μmol/L) for 24 hours in this experiment. The endometrial epithelial cells were from three biologically independent samples, and the data were shown in triplicate.
Total RNAs were isolated using TRIzol reagent (Life Technologies) according to the handbook of the kit. The above RNAs were subjected to mRNA high-throughput sequencing analysis by The Beijing Genomics Institute (BGI). The differential genes were clustered, and the heat map was plotted using the OmicShare tools. Data analysis was performed using the BD CellQuest Pro software.

| Flow cytometry assay
The percentage of cell apoptosis was assessed by flow cytometry assay according to the protocol of Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime) in this experiment. Briefly, cells were washed with cold PBS and re-suspended in the binding buffer at a concentration 1 × 10 6 cells/mL, and each tube of mixture was incubated with Annexin V-FITC and propidium iodide for 15 minutes at room temperature.
Data analysis was performed using the BD Cell Quest Pro software.

| TUNEL
Apoptotic cells in the myometrium of adenomyosis uterus were further detected using a TUNEL colorimetric staining (In situ apoptosis detection kit, Invitrogen) according to the manufacturer's instructions. TUNEL staining was performed with fluorescein-dUTP for apoptotic cell nuclei, and DAPI was used to stain all cell nuclei.
After labelling, the TUNEL-positive cells were visualized and counted under a fluorescence microscopy.

| Quantitative real-time PCR
The protocol of reverse transcription-polymerase chain reaction (RT-PCR) is based on protocol of Transcriptor First Strand cDNA Synthesis Kit (Takara). Total RNA (1 μg) was used as the template for the cDNA synthesis reaction with random primers.

| Immunohistochemical analysis
The sections were from adenomyosis samples, and immunohistochemical assay was performed as previously described. 23 The results were evaluated by two independent investigators using a semi-quantitative scale. A semi-quantitative grading of CXCR4 expression was represented as the sum of percentage score and intensity score. Scores that correspond to the percentages of staining cells were defined as follows: 0 for no documented positive staining cell, 1 for the 25% positive staining cells, 2 for >25% and 50%, and 3 for >50%. Moreover, in terms of intensity of the stain, the following scores were designated as follows: 0 for no documented stains, 1 for a weak, 2 for moderate and 3 for high. 24

| Statistical tests
SPSS program version 19.0 and Graph Pad Prism 5 software were used for statistical analysis. Data are presented as the mean ± Standard Error of Mean (SEM). P values were determined by the two-tailed Student's t test or Mann-Whitney U test when comparing two groups and by a one-way ANOVA when comparing more than two groups. Statistical difference was considered to be significant at a value of P < .05 (*), highly significant at a value of P < .01 (**) and extremely significant when P < .001 (***).

| Mifepristone decreases the viability of endometrial epithelial cells and stromal cells in adenomyosis
To study the effect of mifepristone on the viability of endometrial epithelial cells and stromal cells of adenomyosis, CCK-8 assay was performed. Human primary endometrial epithelial cells and stromal cells were treated with mifepristone in different concentrations (0, 10, 25, 50, 75, 100 and 200 μmol/L, respectively) for 48 hours.
As shown in Figure 1A, the treatment of mifepristone exhibited a concentration-dependent inhibitory effect on both endometrial epithelial and stromal cells compared with the controls. Especially, the viability of endometrial cells was significantly decreased when treated with mifepristone at concentrations above 50 μmol/L at 48 hours. These results indicated that mifepristone inhibits the viability of endometrial epithelial cells and stromal cells in adenomyosis.

| Mifepristone inhibits the migratory capacity of eutopic endometrial epithelial cells and stromal cells in adenomyosis
Migration is a critical step during infiltration of eutopic endometrial epithelial cells and stromal cells into myometrium. To investigate . Data were presented as the mean ± SEM. Conc., concentrations; EEC, endometrial epithelial cells; ESC, endometrial stromal cells. *P < .05, **P < .01, ***P < .001 F I G U R E 2 Mifepristone down-regulates the expressions of CDK1, CDK2, cyclin B, cyclin E and CXCR4 in endometrial epithelial cells by analysis of RNA-Seq data. A, Primary endometrial epithelial cells were treated with mifepristone (0, 50 and 100 μmol/L, respectively) for 24 h (n = 3). Red represents up-regulation and green represents down-regulation. B, KEGG analyse was conducted. C, The expressions of cyclin B, cyclin E, CDK1, CDK2 and CXCR4 were significantly down-regulated in endometrial epithelial cells after treatment with mifepristone when compared to controls. C, Control (C-1, the first patient in control group; C-2, the second patient in control group; C-3, the third patient in control group); T1, cells were treated with mifepristone at 50 μmol/L for 24 h (T1-1, the first patient in T1 group; T1-2, the second patient in T1 group; T1-3, the third patient in T1 group); T2, cells were treated with mifepristone at 100 μmol/L for 24 h (T2-1, the first patient in T2 group; T2-2, the second patient in T2 group; T2-3, the third patient in T2 group) the effects of mifepristone on the migratory capacity of eutopic endometrial epithelial cells and stromal cells in adenomyosis, migration assay was performed. Comparing with plain media controls, the number of migrated endometrial epithelial cells and stromal cells were both increased by FBS attraction in bottom wells.
However, the migratory response of eutopic endometrial epithelial cells and stromal cells was significantly restricted after treatment with mifepristone in a dose-dependent manner ( Figure 1B).
The results demonstrated that mifepristone inhibits the migration capacity of eutopic endometrial epithelial cells and stromal cells in adenomyosis.

| Mifepristone down-regulates the gene expressions of CDK1, CDK2, cyclin B, cyclin E and CXCR4 in endometrial epithelial cells by analysis of RNA-Seq data
To investigate the potential mechanism of mifepristone treatment on the adenomyosis, gene expression was examined in the primary endometrial epithelial cells with or without treatment of mifepristone by RNA sequencing. The cells were treated with mifepristone (0, 50 and 100 μmol/L, respectively) for 24 hours (n = 3). Different genes between control group and mifepristone-treated groups were F I G U R E 3 Mifepristone induces cell cycle arrest of endometrial epithelial and stromal cells through inhibiting CDK1 and CDK2 expressions. A, Cell cycle analysis was performed to quantify the cycle phase distribution of endometrial epithelial and stromal cells after mifepristone treatment. The cells were treated with mifepristone at different concentrations (0, 50, 100 and 200 μmol/L) and subjected to flow cytometric analysis. The results showed that mifepristone increased G2/M and G0/G1 phase cell population but decreased S phase cell population in a dose-dependent manner. B, The down-regulations of CDK1 and CDK2 were further confirmed by qRT-PCR analysis in endometrial epithelial and stromal cells after treatment of mifepristone. C, Protein levels of CDK1, cyclin B and CDK2 and cyclin E were detected by Western blotting analysis in primary endometrial epithelial and stromal cells of adenomyosis after treatment of mifepristone. The band densitometry was quantified using Image J software and relative protein expression was graphed. Protein samples were from three different patients for each group. Data were presented as the mean ± SEM from three biologically independent samples. Conc., concentrations; EEC, endometrial epithelial cells; ESC, endometrial stromal cells. *P < .05, **P < .01, ***P < .001  (Figure 2A). KEGG analyses found that the responses to mifepristone treatment were signal transduction, cell growth and death, cellular community and cell motility, etc ( Figure 2B). Figure 2C showed that mifepristone prominently down-regulated the expressions of CDK1, CDK2, cyclin B, cyclin E and CXCR4 in endometrial epithelial cells of adenomyosis when compared to controls, which are the key genes for regulating cell proliferation, apoptosis and migration.

| Mifepristone induces cell cycle arrest of endometrial epithelial cells and stromal cells through inhibiting CDK1 and CDK2 expressions
It is well known that the cell cycle is regulated by the action of a family of serine/threonine kinases known as cyclin-dependent kinases (CDKs). According to the previous result of RNA sequencing ( Figure 2C), the expressions of CDK1, CDK2, cyclin B and cyclin E were significantly decreased in endometrial epithelial cells treated with mifepristone when compared to control. The down-regulations of CDK1 and CDK2 in the mifepristone-treated group were further confirmed by qRT-PCR analysis not only in primary endometrial epithelial cells but also in stromal cells ( Figure 3B). Additionally, the results of Western blotting were also consistent with RNA sequencing and qRT-PCR analysis ( Figure 3C). Furthermore, this inhibitory effect of mifepristone was in a dose-dependent response. Taken together, it is therefore speculated that mifepristone induces cell cycle arrest of endometrial epithelial cells and stromal cells through inhibiting CDK1 and CDK2 expressions.

| Mifepristone induces apoptosis of endometrial epithelial cells and stromal cells through the mitochondria-dependent signalling pathway
To further validate the mechanism of apoptosis induced by mifepristone in endometrial cells of adenomyosis, Western blotting was performed to detect the effect of mifepristone on the expression of a molecular marker of cell apoptosis. As the drug concentration increased, the expression of caspase-3 protein was mildly decreased, while cleaved caspase-3 was significantly increased in both primary endometrial epithelial cells and stromal cells of adenomyosis ( Figure 4C). Since caspase-3 is a critical mediator of mitochondrial events of cell apoptosis, the data indicated mifepristone induces apoptosis of endometrial epithelial cells and stromal cells through the mitochondria-dependent signalling pathway.

| Mifepristone inhibits the migratory capacity of the eutopic endometrial epithelial cells and stromal cells through suppressing CXCR4 expression in adenomyosis
The result of RNA sequencing showed that mRNA expression of

| Mifepristone inhibits epithelial-mesenchymal transition of endometrial epithelial cells in adenomyosis
Epithelial-mesenchymal transition is a vital step in the invasion and metastasis of endometrial epithelial cells in adenomyosis. In an attempt to investigate the effect of mifepristone on EMT process of eutopic endometrial epithelial cells in adenomyosis, EMT-related markers were checked. The mRNA level of N-cadherin was significantly down-regulated in endometrial epithelial cells when treated with mifepristone in a dose-dependent manner. Similar trend was also found in N-cadherin protein expression detected by Western blotting ( Figure 5D). However, there was no increase in the protein level of E-cadherin in endometrial epithelial cells treated with mifepristone when compared with untreated controls (data were not shown). The findings signified that mifepristone inhibits the EMT process of endometrial epithelial cells in adenomyosis.

| Mifepristone serves as a novel therapeutic drug in the treatment of adenomyosis
It is known that uterine volume is always increased and associated with severity of adenomyosis. To further study the therapeutic efficacy of mifepristone on adenomyosis patients, uterine volume was measured by transvaginal ultrasound (TVS) and MRI in this study.
As indicated in the representative uterine images of TVS and MRI ( Figure 6A), the uterine volume was decreased after three-month treatment with mifepristone in the same adenomyosis patient. As shown in Figure 6B, the mean uterine volume before treatment was 247.7 ± 18.9 mm 3 while the mean volume was 192.7 ± 19.3 mm 3 after three-month treatment based on the TVS measurement, and there is a statistically significant reduction (P < .001). Furthermore, the CA125 and haemoglobin concentrations in serum were two important markers for the development of adenomyosis. The mean CA125 concentration in serum before treatment was 193.8 ± 27.2 U/mL while the mean concentration was 96.6 ± 18.7 U/mL after threemonth treatment, and haemoglobin concentration in serum was increased from 106.9 ± 6.0 to 123.4 ± 4.7 g/L. The results showed that CA125 concentration in serum was dramatically decreased while haemoglobin concentration was significantly increased after mifepristone treatment ( Figure 6B). Our data showed that mifepristone decreases the uterine volume and CA125 concentration while increases haemoglobin concentration in serum for adenomyosis patient and therefore could serve as a novel therapeutic drug in the treatment of adenomyosis.

| D ISCUSS I ON
Adenomyosis is benign invasion of the endometrium into the myometrium, and it shows malignant features, such as abnormal growth, invasion and migration. 25 33 However, there are few studies about the application of mifepristone in adenomyosis patients and its mechanism of therapeutic efficacy. Firstly, we found that mifepristone directly F I G U R E 6 Mifepristone could be applied in the treatment of adenomyosis. A, Representative uterine images of TVS and MRI in the same adenomyosis patient before and after three-month treatment with mifepristone. B, Median uterine volume was calculated in 20 adenomyosis patients before and after mifepristone treatment. Ultrasonography was applied to measuring the length of the long-and shortaxis of the sagittal section and the long-axis of the cross section of the uterus. Uterine volume was assessed using the formula for an ovoid (length × width × depth × 0.52). The CA125 concentration and haemoglobin concentration in serum of adenomyosis patient were measured before and after three-month treatment with mifepristone. MRI, magnetic resonance imaging; TVS, transvaginal ultrasound. Data were expressed as mean ± SEM. *P < .05, **P < .01 and ***P < .001 Epithelial-mesenchymal transition serves as an important role in the development of adenomyosis by promoting cell invasion. 36,37 It is reported that E-cadherin-negative and N-cadherin-positive endometriotic epithelial cells were greatly invasive in vitro. 38 Previous studies found that mifepristone blocks the EMT-related signalling pathways to inhibit the invasion of peritoneal mesothelial cells and endometrial cancer cells. 39 In our study, the results indicated that N-cadherin, a characteristic molecular feature of EMT, was significantly decreased in endometrial epithelial cells treated with mifepristone in a dose-dependent manner. Therefore, mifepristone restricts the invasion of endometrial epithelial cells via suppression of EMT process in adenomyosis.
It is known that large uterine is a feature for the adenomyosis and results from sustained proliferation and continued migration of endometrial cells. Uterine volume is always increased and associated with severity of adenomyosis. Furthermore, CA125 is a traditional marker for the diagnosis and progression of adenomyosis. In this study, we demonstrated that uterine volume and CA125 concentration in serum were significantly decreased after treatment of mifepristone for adenomyosis patients. In addition, hypermenorrhoea always happens and results in anaemia in adenomyosis patients. The results showed that haemoglobin concentration was significantly increased after mifepristone treatment. Therefore, our data proved that mifepristone might serve as a novel therapeutic drug in the treatment of adenomyosis.
In summary, the present study first investigated the direct effects of mifepristone on primary endometrial epithelial cells and stromal cells in adenomyosis. Mifepristone causes cell cycle arrest through inhibiting CDK1 and CDK2 expression and induces cell apoptosis via the mitochondria-dependent signalling pathway in endometrial epithelial cells and stromal cells of adenomyosis. Furthermore, mifepristone inhibits the migration of endometrial epithelial cells and stromal cells through decreasing CXCR4 expression and restricts the invasion of endometrial epithelial cells via suppression of EMT process in adenomyosis (Figure 7). We also report that mifepristone treatment decreases the uterine volume, CA125 concentration and increases haemoglobin concentration in serum for adenomyosis patient. Therefore, we demonstrate that mifepristone could serve as a novel therapeutic drug in the treatment of adenomyosis and the old dog can do a new trick.

CO N FLI C T O F I NTE R E S T
The authors declare no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.