Concentrations Of Mifepristone Research Articles

Mifepristone increases AQP1 mRNA expression, angiogenesis, and cell permeability through the ERK MAPK pathway.

The purpose of this study was to investigate the mechanism of mifepristone serves as an anti-implantation contraceptive drug on aquaporins 1 (AQP1) expression. Human umbilical vein endothelial cells (HUVECs) were used to detect the effects of different concentrations of mifepristone (0, 0.065, 0.2, and 1μmol/L) on the activity of angiogenesis and AQP1 expression. The expression of AQP1 was tested by the real-time PCR. The angiogenesis and penetration function of HUVECs was investigated by Matrigel lumen formation and trans-well assay, respectively. The expression of AQP1, angiogenesis and cell permeability were significantly higher than control groups in HUVECs treatment with mifepristone at 1μmol/L for 12h. Estrogen and progesterone decreased the up-regulation of AQP1 and cell permeability, not angiogenesis, induced by mifepristone. Mifepristone increased protein levels of p-ERK, not p-p38 or p-JNK, and pre-treatment with ERK MAPK-specific inhibitor significantly inhibited the up-regulation of AQP1 mRNA expression, angiogenesis and cell permeability induced by mifepristone. si-AQP1 significantly reduced the up-regulation of angiogenesis, cell permeability and p-ERK/ERK ratio expression induced by mifepristone treatment. Overexpression of AQP1 enhanced the increase of expression ratio of p-ERK/ERK induced by mifepristone. Low-dose mifepristone increased cell permeability, angiogenesis and AQP1 expression, which was involved in MAPK pathways. This provides new insights into the molecular mechanism of mifepristone serves as an anti-implantation contraceptive drug.

Determination of Mifepristone (RU-486) and Its Metabolites in Maternal Blood Sample after Pharmacological Abortion.

The aim of the study was the development and validation of the UHPLC-QqQ-MS/MS method for the determination of mifepristone in human blood as well as the identification and quantification of its metabolites after self-induced pharmacological abortion. The metabolic pathway in humans was proposed after examination of an authentic casework. The fast and simple preanalytical procedure was successfully applied (pH9, tert-butyl-methyl ether). The validation parameters of the method were as follows: limit of quantification: 0.5 ng/mL; coefficients of determination: >0.999 (R2), intra- and inter-day accuracy and precision values did not exceed ± 13.2%. The recovery and matrix effect were in the range of 96.3-114.7% and from -3.0 to 14.7%, respectively. Toxicological analysis of the mother's blood (collected the day after the pregnancy termination) revealed the presence of five compounds: mifepristone (557.4 ng/mL), N-desmethyl-mifepristone (638.7 ng/mL), 22-OH-mifepristone (176.9 ng/mL), N,N-didesmethyl-mifepristone (144.5 ng/mL) and N-desmethyl-hydroxy-mifepristone (qualitatively). To our knowledge, the study presented in this paper is the first report on the concentrations of mifepristone and its metabolites in maternal blood samples after performing a self-induced abortion. The established UHPLC-QqQ-MS/MS method is suitable for forensic toxicological analysis as well as in terms of clinical toxicology in future investigations (examination of pharmacokinetics, bioavailability and metabolism of RU-486).

Anticancer effects of mifepristone on human uveal melanoma cells

BackgroundUveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds.MethodsThe effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR.ResultsMF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor.ConclusionThis study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

Mifepristone regulates macrophage-mediated natural killer cells function in decidua

Mifepristone has been used for first-trimester abortion and contraception. Nevertheless, its functional mechanism still needs to be elucidated. Decidua tissues were collected from 40 pregnant women who received (20 patients) or did not receive (20 patients) mifepristone. Immunofluorescence was used to analyze the effect of mifepristone on the quantity of CD56 and CD206 in decidua. in vitro assay, NK cells were isolated from decidua tissue and macrophages were induced from THP-1 cells. NK cells were co-cultured with macrophages pre-treated different concentrations of mifepristone (0 nmol/L, 200 nmol/L, 1800 nmol/L, and 25000 nmol/L); the cells' cytotoxicity and migration ability were analyzed using MTT assay and transwell assay, respectively. Si-TGF-β1, which was utilized to knock down the TGF-β1 expression in macrophages and human recombinant TGF-β1 were used to verify whether TGF-β1 was involved in the mifepristone regulation of NK cells function. The quantity of CD56 and CD206 decreased after mifepristone treatment. Moreover, the NK cells' cytotoxicity and migration ability were significantly increased by macrophages pre-treated with mifepristone in a dose-dependent manner. Moreover, compared with the si-NC group, the MTT absorbance rate of NK cells was significantly increased in the si-TGF-β1 group and was decreased in the human recombinant TGF-β1 group. Our data suggest that mifepristone, which regulates NK cells function through macrophages, was associated with the changes in TGF-β1 secreted by macrophages. This may be one of the mechanisms of mifepristone acting as contraceptive and abortion drugs at the maternal-fetal interface.

Mifepristone regulates Tregs function mediated by dendritic cells through suppressing the expression of TGF-β

Background Previous studies have demonstrated that mifepristone in the daily low-dose affects the function of endometrium. These researches also implied an alteration of endometrium immune balance, which might be involved in regulating endometrial function. However, the detailed mechanisms remain to be further explored. Methods In this study, the expressions of CD80, CD86, and ICAM-1 in dendritic cells (DCs), which were stimulated with different concentrations of mifepristone (20, 65, and 200 nM), were detected by FACS. After that, we further evaluated the expression of Forkhead box P3 (FOXP3) and IL-10 in Tregs, which co-cultured with mifepristone treated DCs. In mechanism, we compared the indoleamine 2,3-dioxygenase (IDO) and TGF-β expression with enzyme-linked immunosorbent assay (ELISA). Results The results indicated that mifepristone promoted the expressions of CD80, CD86, and ICAM-1 in a dosage dependent manner. Reversely, FOXP3 and IL-10 expression levels in Tregs co-cultured with mifepristone-treated DCs were significantly decreased compared with those co-cultured with nontreated DC. Furthermore, a significant reduce in IDO and TGF-β expression was observed in DCs treated with mifepristone. By using the IDO inhibitor (1-methyl tryptophan, 1-MT) or TGF-b supplement, we confirmed that TGF-β, but not IDO could rescue the downregulation of FOXP3 and IL-10 in Tregs co-cultured with mifepristone treated DCs. All of these results suggest that mifepristone may regulate DC function by decreasing TGF-β expression, which further results in the downregulations of FOXP3 and IL-10 in Tregs. Conclusion Therefore, our research provides a theoretical basis for a potentially clinical application of mifepristone as a novel contraceptive.

Effects of progesterone receptor on proliferation of uterine leiomyoma cells.

In order to study the mechanism of the effect of progesterone receptor on the growth of primary uterine leiomyoma cells, the primary cells were extracted from uterine leiomyoma cells and identified by immunohistochemistry (IHC). Mitochondrial progesterone receptor-positive [PR-M(+)], mitochondrial progesterone receptor-negative [PR-M(-)], progesterone receptor A (PR-A) and progesterone receptor B (PR-B) were screened by Western blotting. Different concentrations of Mifepristone (MIF), a progesterone receptor antagonist, were used to interfere with PR-M(+) and PR-M(-) cell lines, respectively. Proliferation and apoptosis of PR-M(+) and PR-M(-) cell lines were detected by tetramethylazolyl blue method and flow cytometry, respectively. The expression of Caspase-3 and B-cell lymphoma 2 (Bcl-2) protein was detected by Western blotting. The results showed that the growth of PR-M(+) and PR-M(-) uterine leiomyoma cells was inhibited with the increase of MIF concentration. Furthermore, the proliferation inhibition rate and apoptosis rate were gradually increased. However, the expression of Caspase-3 protein on progesterone receptor M increased, while the expression of Bcl-2 decreased. Moreover, progesterone could induce progesterone receptor M to up-regulate apoptotic protein Caspase-3 and down-regulate anti-apoptotic protein Bcl-2, thus it could inhibit the apoptosis of primary cultured uterine leiomyoma cells and promote the proliferation of leiomyoma cells.

Advanced assessment of migration and invasion of cancer cells in response to mifepristone therapy using double fluorescence cytochemical labeling

BackgroundPrevious work in our laboratory demonstrated that antiprogestin mifepristone impairs the growth and adhesion of highly metastatic cancer cells, and causes changes in their cellular morphology. In this study, we further assess the anti-metastatic properties of mifepristone, by studying whether cytostatic doses of the drug can inhibit the migration and invasion of various cancer cell lines using a double fluorescence cytochemical labeling approach.MethodsCell lines representing cancers of the ovary (SKOV-3), breast (MDA-MB-231), glia (U87MG), or prostate (LNCaP) were treated with cytostatic concentrations of mifepristone. Wound healing and Boyden chamber assays were utilized to study cellular migration. To study cellular invasion, the Boyden chamber assay was prepared by adding a layer of extracellular matrix over the polycarbonate membrane. We enhanced the assays with the addition of double fluorescence cytochemical staining for fibrillar actin (F-actin) and DNA to observe the patterns of cytoskeletal distribution and nuclear positioning while cells migrate and invade.ResultsWhen exposed to cytostatic concentrations of mifepristone, all cancer cells lines demonstrated a decrease in both migration and invasion capacities measured using standard approaches. Double fluorescence cytochemical labeling validated that mifepristone-treated cancer cells exhibit reduced migration and invasion, and allowed to unveil a distinct migration pattern among the different cell lines, different arrays of nuclear localization during migration, and apparent redistribution of F-actin to the nucleus.ConclusionThis study reports that antiprogestin mifepristone inhibits migration and invasion of highly metastatic cancer cell lines, and that double fluorescence cytochemical labeling increases the value of well-known approaches to study cell movement.

Effects of progesterone receptor on proliferation of uterine leiomyoma cells.

In order to study the mechanism of the effect of progesterone receptor on the growth of primary uterine leiomyoma cells, the primary cells were extracted from uterine leiomyoma cells and identified by immunohistochemistry (IHC). Mitochondrial progesterone receptor-positive [PR-M(+)], mitochondrial progesterone receptor-negative [PR-M(-)], progesterone receptor A (PR-A) and progesterone receptor B (PR-B) were screened by Western blotting. Different concentrations of Mifepristone (MIF), a progesterone receptor antagonist, were used to interfere with PR-M(+) and PR-M(-) cell lines, respectively. Proliferation and apoptosis of PR-M(+) and PR-M(-) cell lines were detected by tetramethylazolyl blue method and flow cytometry, respectively. The expression of Caspase-3 and B-cell lymphoma 2 (Bcl-2) protein was detected by Western blotting. The results showed that the growth of PR-M(+) and PR-M(-) uterine leiomyoma cells was inhibited with the increase of MIF concentration. Furthermore, the proliferation inhibition rate and apoptosis rate were gradually increased. However, the expression of Caspase-3 protein on progesterone receptor M increased, while the expression of Bcl-2 decreased. Moreover, progesterone could induce progesterone receptor M to up-regulate apoptotic protein Caspase-3 and down-regulate anti-apoptotic protein Bcl-2, thus it could inhibit the apoptosis of primary cultured uterine leiomyoma cells and promote the proliferation of leiomyoma cells.

Low-dose mifepristone increased angiogenesis in a manner involving AQP1.

To investigate the molecular mechanisms governing aquaporin-1 (AQP1)-mediated, mifepristone-induced angiogenesis and improve the understanding of low-dose mifepristone serving as an anti-implantation contraceptive drug. Human umbilical vein endothelial cells (HUVECs) were used to explore the effects of different concentrations of mifepristone (0, 65, and 200nmol/L) on the activity of angiogenesis. Forty-five pregnant mice during the "window of implantation" were treated with different concentrations of mifepristone. HUVECs' proliferation was examined using a methyl thiazolyl tetrazolium (MTT) assay. The microvessel density (MVD) and the expression of AQP1 in endometrium were determined with immunohistochemical methods. The MVD and the expression of AQP1 were significantly higher than controls. Mifepristone at 200nmol/L significantly affected HUVECs' proliferation during culture over 12h, and pretreatment with AQP1-specific siRNA significantly inhibited the mifepristone-enhanced cell proliferation. Low-dose mifepristone increased angiogenesis in a manner involving AQP1. This affords a new insight into the molecular mechanism underpinning the angiogenic effects of low-dose mifepristone.

Effect of mifepristone on human sperm function in vitro

Objective: Mifepristone, as a type of well-accepted emergency contraceptive drugs, is an optional progesterone receptor modulator and widely used clinically. However, the effect of mifepristone on sperms is poorly studied. This research is intended to investigate the biological effect of mifepristone on human sperm function in vitro. Methods: Spermatozoa were incubated in the culture medium containing 0.04 μM, 0.4 μM, 4 μM and 40 μM of mifepristone for one hour after capacitation. It was required to measure sperm motility, viability, DNA integrity, acrosomal reaction, sperm hyperactivation, and the concentration of free calcium ions in spermatozoa. Results: After treating sperms with different concentrations of mifepristone in vitro, sperm viability, motility, morphology and DNA integrity were measured. The differences between the control group and the experimental group were of no statistical significance ( p > .05). When the concentration of mifepristone was higher than 0.4 μM, the proportion of sperm hyperactivation was significantly reduced ( p < .01). According to further examination on the rate of acrosomal reaction (AR rate) and the concentration of free calcium ions in sperms, it was found that acrosomal reaction was significantly inhibited with the concentration of free calcium ions in sperms decreased when the concentration of mifepristone was higher than 0.4 μM. Conclusions: The in-vitro spermicidal mechanism of mifepristone is realized by inhibiting acrosomal reaction, hyperactivation and decreasing the concentration of free calcium ions.

Effect of mifepristone on human sperm function in vitro

Objective: Mifepristone, as a type of well-accepted emergency contraceptive drugs, is an optional progesterone receptor modulator and widely used clinically. However, the effect of mifepristone on sperms is poorly studied. This research is intended to investigate the biological effect of mifepristone on human sperm function in vitro. Methods: Spermatozoa were incubated in the culture medium containing 0.04 μM, 0.4 μM, 4 μM and 40 μM of mifepristone for one hour after capacitation. It was required to measure sperm motility, viability, DNA integrity, acrosomal reaction, sperm hyperactivation, and the concentration of free calcium ions in spermatozoa. Results: After treating sperms with different concentrations of mifepristone in vitro, sperm viability, motility, morphology and DNA integrity were measured. The differences between the control group and the experimental group were of no statistical significance ( p &gt; .05). When the concentration of mifepristone was higher than 0.4 μM, the proportion of sperm hyperactivation was significantly reduced ( p &lt; .01). According to further examination on the rate of acrosomal reaction (AR rate) and the concentration of free calcium ions in sperms, it was found that acrosomal reaction was significantly inhibited with the concentration of free calcium ions in sperms decreased when the concentration of mifepristone was higher than 0.4 μM. Conclusions: The in-vitro spermicidal mechanism of mifepristone is realized by inhibiting acrosomal reaction, hyperactivation and decreasing the concentration of free calcium ions.

Mifepristone inhibits proliferation, migration and invasion of HUUA cells and promotes its apoptosis by regulation of FAK and PI3K/AKT signaling pathway.

PurposeThe aim was to investigate mifepristone effects on endometrial carcinoma and the related mechanism.MethodsHHUA cells were treated with DMEM containing different concentrations of mifepristone. HHUA cells treated with 100 μmol/L mifepristone were named the Mifepristone group. HHUA cells co-transfected with pcDNA3.1-PI3K and pcDNA3.1-AKT overexpression vectors were treated with 100 μmol/L mifepristone and named the Mifepristone + PI3K/AKT group. mRNA expression was detected by quantitative reverse transcription PCR. Protein expression was performed by Western blot. Cell proliferation was conducted by MTT assay. Wound-healing assay was conducted. Transwell was used to detect cells migration and invasion. Apoptosis detection was performed by flow cytometry.ResultsMifepristone inhibited HHUA cells proliferation in a dose-dependent manner. Compared with HHUA cells treated with 0 μmol/L mifepristone, HHUA cells treated by 50–100 μmol/L mifepristone had a lower wound-healing rate, a greater number of migrating and invasive cells (P<0.01), as well as a higher percentage of apoptotic cells and Caspase-3 expression (P<0.01). When HHUA cells were treated with 50–100 μmol/L of mifepristone, FAK, p-FAK, p-PI3K and p-AKT relative expression was all significantly lower than HHUA cells treated with 0 μmol/L of mifepristone (P<0.01). Compared with the Mifepristone group, HHUA cells of the Mifepristone + PI3K/AKT group had a lower cell growth inhibition rate and percentage of apoptotic cells (P<0.01).ConclusionMifepristone inhibited HUUA cells proliferation, migration and invasion and promoted its apoptosis by regulation of FAK and PI3K/AKT signaling pathway.

Pharmacokinetic differences of mifepristone between sexes in animals

Mifepristone (RU486) is developed originally as a contraceptive used by hundreds of millions of women world-wide, and also reported as a safe and long-term psychotic depressant, or as a cancer chemotherapeutic agent used by both sexes. In our preliminary study aimed at developing mifepristone as a cancer metastatic chemopreventive, we coincidentally observed that blood mifepristone concentrations in female rats seem to be higher than those in male ones post administration. To substantiate if the pharmacokinetic differences between sexes exist, we established a fast UPLC–MS/MS method to determine mifepristone concentrations in plasma, and analyzed blood concentrations of mifepristone over time in rats and dogs of both sexes. Mifepristone in plasma or incubation liquid was recovered by liquid-liquid extraction using 1mL of ethyl acetate. Chromatographic separation was performed on a C18 column at 35°C, with a gradient elution consisting of methanol and water containing 0.1% (v/v) formic acid at a flow rate of 0.3mL/min. And pharmacokinetic parameters such as elimination half-life, and mean residence time were calculated by using the non-compartmental pharmacokinetics data analysis software. In this work, administrations of mifepristone to rats and beagle dogs revealed that the plasma concentrations of mifepristone (AUC, Cmax) were significantly higher (P<0.05) in females than that in males. In vitro liver microsomal incubation experiments showed that the metabolic rate of mifepristone in males was higher than that in females, which was consistent with the results of in vivo experiments. In general, we first found the sex-related differences about pharmacokinetic properties of mifepristone and revealed the metabolism difference of hepatic microsomal enzyme is the main reason.

Mifepristone inhibites the migration of endometrial cancer cells through regulating H19 methylation

To investigate the effect and mechanism of mifepristone on the migration of human endometrial carcinoma cells. A human endometrial carcinoma cell line, Ishikawa cells, was cultured in vitro and treated with mifepristone at different concentrations. Wound healing assay was applied to detect the migration of Ishikawa cells. RT-PCR and methylation-specific PCR (MSP) were used to detect the levels of H19 mRNA and its DNA methylation. Western-blot was used to detect the expressions of HMGA2 and epithelial to mesenchymal transition (EMT) related proteins. When treated with different concentrations of mifepristone for 48 hours, the width of scratch of the the control group, the 5 mg/L and the 10 mg/L mifepristone treatment groups were (4.18±0.07)mm, (4.68±0.07)mm, and(4.99±0.07)mm, respectively (P<0.05 for all) and treated with mifepristone for 72 hours, those were(3.46±0.07)mm, (4.29±0.07)mm, and(4.78±0.04)mm, respectively (P<0.05 for all). In the Ishikawa cells, mifepristone suppressed the transcriptional level of H19 through enhancing its promoter methylation, which resulted in inhibited expressions of HMGA2 and vimentin and increased expression of E-cadherin in a time- and concentration-dependent manner. Mifepristone inhibits the migration of endometrial carcinoma cells partially through methylation-induced of transcriptional inhibition of H19, which results in the down-regulation of HMGA2 and vimentin and upregulation of E-cadherin.

Glucocorticoid receptor antagonism as a novel therapy for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) accounts for 10% to 20% of newly diagnosed invasive breast cancer. Finding effective targets for chemotherapy-resistant TNBC has proven difficult in part because of TNBC's molecular heterogeneity. We have previously reported that likely because of the antiapoptotic activity of glucocorticoid receptor (GR) in estrogen receptor (ER)-negative breast epithelial and cancer cells, high GR expression/activity in early-stage TNBC significantly correlates with chemotherapy resistance and increased recurrence. We hypothesized that pretreatment with mifepristone, a GR antagonist, would potentiate the efficacy of chemotherapy in GR+ TNBCs by inhibiting the antiapoptotic signaling pathways of GR and increasing the cytotoxic efficiency of chemotherapy. TNBC cell apoptosis was examined in the context of physiologic glucocorticoid concentrations, chemotherapy, and/or pharmacologic concentrations of mifepristone. We used high-throughput live microscopy with continuous recording to measure apoptotic cells stained with a fluorescent dye and Western blot analysis to detect caspase-3 and PARP cleavage. The effect of mifepristone on GR-mediated gene expression was also measured. TNBC xenograft studies were performed in female severe combined immunodeficient (SCID) mice and tumors were measured following treatment with vehicle, paclitaxel, or mifepristone/paclitaxel. We found that although mifepristone treatment alone had no significant effect on TNBC cell viability or clonogenicity in the absence of chemotherapy, the addition of mifepristone to dexamethasone/paclitaxel treatment significantly increased cytotoxicity and caspase-3/PARP cleavage. Mifepristone also antagonized GR-induced SGK1 and MKP1/DUSP1 gene expression while significantly augmenting paclitaxel-induced GR+ MDA-MB-231 xenograft tumor shrinkage in vivo. These results suggest that mifepristone pretreatment could be a useful strategy for increasing tumor cell apoptosis in chemotherapy-resistant GR+ TNBC.

Abstract A23: Induction of the unfolded protein response in ovarian cancer cells exposed to cytostatic concentrations of antiprogestin/antiglucocorticoid mifepristone

Abstract Ovarian cancer is rare; yet, it is the deadliest gynecologic malignancy with a 5-year survival rate that has only improved 9% in the past 30 years and a dismal overall survival rate that has remained stagnant for over 50 years. Although most patients diagnosed with advanced disease undergo remission after optimal surgical cytoreduction and platinum (Pt)-based chemotherapy, microscopic foci of cells defined as minimal residual disease (MRD) manage to survive within the peritoneal cavity and recreate the disease (recurrence) that develops a more aggressive phenotype for which current therapies almost always fail. Thus, it is critical to take advantage of the time the patient is in remission to attack the cells constituting the MRD by disengaging their repopulation capacity. Evidence suggests the feasibility of this consolidation therapeutic approach using cytostatic agents. Previously, we demonstrated that the repopulation of cells escaping Pt or Pt-taxane chemotherapy can be abrogated using steroids with antiprogestin/antiglucocorticoid activity, such as mifepristone (MF). We found that MF (i) blocks ovarian cancer cell growth by inducing G1 cell cycle arrest mediated by the up-regulation of cyclin dependent kinase inhibitors p21 and p27, leading to the blockage of the activity of cyclin dependent kinase 2 (Cdk2) and, consequently, lack of DNA synthesis and S phase progression; (ii) induces cell death in combination with blockage of the PI3K/Akt survival pathway; and (iii) it does not require progesterone receptors to exert its anti-growth effect. To further elucidate the cytotoxic mechanism triggered by MF in ovarian cancer cells, we performed genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to Pt. We exposed OV2008 (wt p53, Pt sensitive), OV2008/C13 (wt p53/Pt resistant), or SK-OV-3 (null p53, Pt semi-resistant) cells to cytostatic concentrations of MF for 24, 48 or 72 h. Total RNAs and whole protein extracts were subjected to cDNA microarray analysis and two-dimensional nano-liquid chromatography (2D-nanoLC)-mass spectrometry (MS/MS), respectively. The combined use of cDNA microarray and 2D nanoLC-MS/MS to discover mRNAs and proteins differentially regulated by MF led to the identification of key proteins involved in the regulation of the unfolded protein response (UPR), which were further validated by Western blotting. We identified the master ER chaperone glucose regulated protein (GRP) 78 (also referred to as BiP/HSPA5) highly up-regulated by MF. Also the ER stress sensors protein kinase RNA-like ER kinase (PERK) and inositol-requiring kinase 1 (IRE1alpha) were up-regulated by MF. Activation of PERK and IRE1alpha signaling pathway usually leads to attenuated global protein synthesis thus contributing to cell cycle arrest. We found the translation repressor protein 4E-BP1 augmented by MF suggesting reduced protein translation capability. We also found highly up-regulated the transcription factor C/EBP homologous protein (CHOP), the induction of which is usually linked to cell death. In summary, we identified the UPR as a target for MF activity. We hypothesize that the UPR integrates the cytotoxicity of MF towards ovarian cancer cells when used as monotherapy or in combination therapies triggering, respectively, cell cycle arrest (cytostasis) or cell death (lethality) depending on the degree of cellular stress generated. Citation Format: Maria B. Hapon, Carlos D. Gamarra-Luques, Alicia A. Goyeneche, Eduardo A. Callegari, Kathleen M. Eyster, Carlos M. Telleria. Induction of the unfolded protein response in ovarian cancer cells exposed to cytostatic concentrations of antiprogestin/antiglucocorticoid mifepristone. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A23.

Trough Plasma Concentrations of Mifepristone Correlate with Psychotic Symptom Reductions: A Review of Three Randomized Clinical Trials

Background: The relationship between plasma concentrations of psychiatric medications and their efficacy is rarely evaluated or relied upon in clinical practice. While it is generally accepted in clinical settings that patients respond to varying doses of medication, few studies have formally tested whether plasma concentrations of psychiatric medicine predict patients’ responses. We hypothesized that the amount of mifepristone detected in the plasma after dosing for 7 days would correlate with reduction of psychotic symptoms in patients with psychotic depression. This hypothesis was generated from the exploratory analysis phase of an early clinical trial. In that trial, a statistically significant linear correlation was observed between change in psychotic symptoms and mifepristone plasma concentrations observed at day 7. Method: Secondary analyses were conducted using data from three multicenter randomized clinical trials. For each of the three trials, hypothesis testing was conducted to test whether patients assigned to mifepristone whose plasma concentrations were above pre-specified concentration values would exhibit significantly greater reduction in psychotic symptoms than patients assigned to placebo. Results: In all studies, there was a statistically significant linear correlation between plasma concentration and change in psychotic symptoms. In two of the three clinical trials, the response rate of patients with mifepristone plasma concentrations above 1660 ng/ml was significantly greater than the placebo response rate. Results are presented for the three studies separately and for the combined data. Discussion: Although correlations between plasma correlations and clinical response are not often observed in psychiatric medications, there is evidence that trough mifepristone plasma concentrations are associated with clinical improvement. Study identification numbers clinicaltrials.gov: NCT00130676 (Study 07), NCT00146523 (Study 09), NCT00128479 (Study 06).

Cytostasis and morphological changes induced by mifepristone in human metastatic cancer cells involve cytoskeletal filamentous actin reorganization and impairment of cell adhesion dynamics

BackgroundChanges in cell shape and plasticity in cytoskeletal dynamics are critically involved in cell adhesion, migration, invasion and the overall process of metastasis. Previous work in our laboratory demonstrated that the synthetic steroid mifepristone inhibited the growth of highly metastatic cancer cells, while simultaneously causing striking changes in cellular morphology. Here we assessed whether such morphological alterations developed in response to cytostatic concentrations of mifepristone are reversible or permanent, involve rearrangement of cytoskeletal proteins, and/or affect the adhesive capacity of the cells.MethodsCancer cell lines of the ovary (SKOV-3), breast (MDA-MB-231), prostate (LNCaP), and nervous system (U87MG) were exposed to cytostatic concentrations of mifepristone and studied by phase-contrast microscopy. The transient or permanent nature of the cytostasis and morphological changes caused by mifepristone was assessed, as well as the rearrangement of cytoskeletal proteins. De-adhesion and adhesion assays were utilized to determine if mifepristone-arrested and morphologically dysregulated cells had abnormal de-adhesion/adhesion dynamics when compared to vehicle-treated controls.ResultsMifepristone-treated cells displayed a long, thin, spindle-like shape with boundaries resembling those of loosely adhered cells. Growth arrest and morphology changes caused by mifepristone were reversible in SKOV-3, MDA-MB-231 and U87MG, but not in LNCaP cells that instead became senescent. All cancer cell types exposed to mifepristone displayed greatly increased actin ruffling in association with accelerated de-adhesion from the culture plate, and delayed adhesion capacity to various extracellular matrix components.ConclusionsCytostatic concentrations of mifepristone induced alterations in the cellular structure of a panel of aggressive, highly metastatic cancer cells of different tissues of origin. Such changes were associated with re-distribution of actin fibers that mainly form non-adhesive membrane ruffles, leading to dysregulated cellular adhesion capacity.

Synergistic Lethality of Mifepristone and LY294002 in Ovarian Cancer Cells

We have previously shown that the antiprogestin and antiglucocorticoid mifepristone inhibits the growth of ovarian cancer cells. In this work, we hypothesized that cellular stress caused by mifepristone is limited to cytostasis and that cell killing is avoided as a consequence of the persistent activity of the PI3K/Akt survival pathway. To investigate the role of this pathway in mifepristone-induced growth inhibition, human ovarian cancer cells of various histological subtypes and genetic backgrounds were exposed to cytostatic doses of mifepristone in the presence or absence of the PI3K inhibitor, LY294002. The activation of Akt in ovarian cancer cells, as marked by its phosphorylation on Ser473, was not modified by cytostatic concentrations of mifepristone, but it was blocked upon treatment with LY294002. The combination mifepristone/LY294002, but not the individual drugs, killed ovarian cancer cells via apoptosis, as attested by genomic DNA fragmentation and cleavage of caspase-3, and the concomitant downregulation of antiapoptotic proteins Bcl-2 and XIAP. From a pharmacological standpoint, when assessing cell growth inhibition using a median-dose analysis algorithm, the interaction between mifepristone and LY294002 was synergistic. The lethality caused by the combination mifepristone/LY294004 in 2-dimensional cell cultures was recapitulated in organized, 3-dimensional spheroids. This study demonstrates that mifepristone and LY294002 when used individually cause cell growth arrest; yet, when combined, they cause lethality.

A study of mifepristone/IFN-&amp;gamma;-induced apoptosis of human cholangiocarcinoma cell line FRH-0201 in vitro

ObjectiveTo investigate the effects of mifepristone, a progesterone receptor (PR) antagonist, through the proliferation of human cholangiocarcinoma cell line FRH-0201 in vitro and the possible mechanisms involved.MethodsA two-step addition of poly-HRP anti-mouse immunoglobulin G detection system was used to detect the expression of PR in FRH-0201 cells. After treatments with various concentrations of mifepristone (10, 20, 40, 80, 160, and 320 μmol/L) at various time intervals (24, 48, and 72 hours), the rate of cell inhibition, the rate of cell apoptosis, and the expression of bax/bcl-2/Fas were analyzed with tetrazolium blue (MTT) assay, flow cytometry, reverse transcription polymerase chain reaction and Western blotting. The effect of mifepristone and mifepristone combined with interferon (IFN)-γ-inducing apoptosis on the cells was observed.ResultsMifepristone remarkably inhibited the proliferation of FRH-0201 cells, which was revealed by MTT assay in a dose- and time-dependent manner. The inhibitory rate gradually increased following the increase of the dosage of mifepristone from a low dosage (10 μmol/L) to a high dosage (320 μmol/L) at different time intervals. Flow cytometry analysis showed mifepristone increased the rate of the FRH-0201 cell-line apoptosis. Notably, the rate of apoptosis increased markedly when the cells were pretreated with IFN-γ and then treated with mifepristone. In addition, mifepristone obviously upregulated bax and Fas expression and downregulated bcl-2 expression.ConclusionMifepristone effectively inhibited the growth of PR-positive human cholangiocarcinoma cell line FRH-0201 in vitro through multiple mechanisms. Mifepristone combined with IFN-γ might therefore induce the apoptosis of the cell line, which is possibly a beneficial clinical scheme for patients suffering from cholangiocarcinoma.