{"paper_id":"5e8e70d5-8232-40c8-96c9-0dd55d5c4917","body_text":"Combined treatment with U0126 and MK2206 produced a synergic effect in DES grown on substrates of varying stiffness (2- or 30-kPa PGS, or plastic) for ED 95 (effect dose at which 95% growth inhibition occurs), ED 90, and ED 75 (See  Supplementary Fig. S1  &  Supplementary Table S1 ). For ED 50, when DES were grown on 30-kPa PGS or plastic, an additive or an antagonistic effect was produced, whereas in cells grown on 2-kPa PGS, a synergic effect was observed (See  Supplementary Fig. S1  &  Supplementary Table S1 ). In EES derived from the proliferative phase (EES-P), EES derived from the secretory phase (EES-S) and EES derived from the menstrual phase (EES-M), combined U0126 and MK2206 treatment produced an additive or antagonistic effect in cells grown on substrates of varying stiffness (2- or 30-kPa PGS, or plastic) (See  Supplementary Fig. S1  &  Supplementary Table S1 ).\nNo significant differences in cell proliferation were observed among DES, EES-P, EES-S, and EES-M compared to cells grown on a substrate of the same stiffness (2- or 30-kPa PGS, or plastic) at both higher (U0126 [30 μM] and MK2206 [9 μM]) and lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined doses ( Fig. 1 ).\nNo significant differences in cell proliferation were observed between EES and NEES (EES-P versus NEES-P, EES-S versus NEES-S, or EES-M versus NEES-M) when compared to cells grown on a substrate of the same stiffness (2- or 30-kPa PGS, or plastic) at both higher (U0126 [30 μM] and MK2206 [9 μM]) and lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined doses (See  Supplementary Fig. S2 ).\nIn DES ( Fig. 2 ), cell proliferation was significantly more inhibited in cells grown on plastic than those grown on 2-kPa or 30-kPa PGS, when cells were treated with a higher (U0126 [30 μM] and MK2206 [9 μM]) combined dose. However, no significant effects of substrates of varying stiffness (2- or 30-kPa PGS, or plastic) on cell proliferation of DES were observed when cells were treated with a lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined dose ( Fig. 2 ). In EES-P, EES-S, NEES-P and NEES-S, cell proliferation was significantly more inhibited in cells grown on plastic or 30-kPa PGS compared to those grown on 2-kPa PGS when cells were treated with a higher combined dose (U0126 [30 μM] and MK2206 [9 μM]) and/or a lower combined dose (U0126 [15 μM] and MK2206 [4.5 μM]) (See  Supplementary Fig. S3 ). No significant effect of substrates of varying stiffness (2- or 30-kPa PGS, or plastic) was observed on cell proliferation of either EES-M or NEES-M (See  Supplementary Fig. S3 ) treated with either a higher (U0126 [30 μM] and MK2206 [9 μM]) or lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined dose.\nThe percentage of Annexin V-positive cells treated with U0126 alone was significantly higher in DES, EES-S, and EES-M compared to that in EES-P ( Fig. 3 ). When cells were treated with MK2206 alone, the percentage of Annexin V-positive cells was significantly higher in EES-M compared to that in DES, EES-P, and EES-S ( Fig. 3 ). When cells were treated with combination U0126 and MK2206, the percentage of Annexin V-positive cells was significantly higher in DES compared to that in EES-P, EES-S, and EES-M ( Fig. 3 ). In addition, the percentage of Annexin V-positive cells was significantly higher in EES-S and EES-M compared to that in EES-P treated with combination U0126 and MK2206 ( Fig. 3 ).\nSA-βgal activity was observed in DES and EES-P treated with MK2206 alone ( Fig. 4A ). Levels of cyclin D1 mRNA were significantly higher in both DES and EES-P treated with MK2206 alone compared to the vehicle-treated control ( Fig. 4B,C ). Levels of p53 and p21 mRNAs of DES and those of p21 mRNA in EES-P were significantly higher in cells treated with U0126 alone, MK2206 alone, or combination U0126 and MK2206 compared to cells treated with vehicle alone ( Fig. 4B,C ).\nWhen cells were grown on 2-kPa PGS, cell proliferation of EES-M after a 72-h drug discontinuation was significantly higher than that of EES-S at a higher (U0126 [30 μM] and MK2206 [9 μM]) combined dose ( Fig. 5 ). When cells were grown on 30-kPa PGS or plastic, cell proliferation after a 72-h drug discontinuation was significantly higher in DES compared to that of EES-P, EES-S, and EES-M at a lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined dose and compared to that of EES-P and EES-S at a higher (U0126 [30 μM] and MK2206 [9 μM]) combined dose ( Fig. 5 ). In addition, cell proliferation of EES-M grown on 30-kPa PGS or plastic was significantly higher than that of EES-S after a 72-h drug discontinuation of a higher (U0126 [30 μM] and MK2206 [9 μM]) combined dose ( Fig. 5 ).\nWhen a lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined dose was applied, no significant difference in cell proliferation was observed after a 72-h drug discontinuation between EES and NEES (EES-P versus NEES-P, EES-S versus NEES-S, or EES-M versus NEES-M) compared to cells grown on a substrate of the same stiffness (2- or 30-kPa PGS, or plastic) (See  Supplementary Fig. S4 ). However, when a higher (U0126 [30 μM] and MK2206 [9 μM]) combined dose was applied, cell proliferation after a 72-h drug discontinuation was significantly higher in EES-P than in NEES-P when cells were grown on 2-kPa PGS, and significantly higher in EES-M than in NEES-M when compared to cells grown on a substrate of the same stiffness (2- or 30-kPa, or plastic) (See  Supplementary Fig. S4 ). No significant difference in cell proliferation after a 72-h drug discontinuation was observed between EES-S and NEES-S when compared to cells grown on a substrate of the same stiffness (2- or 30-kPa PGS, or plastic) (See  Supplementary Fig. S4 ).\nCell proliferation of DES was significantly higher in cells grown on 30-kPa PGS and plastic than those grown on 2-kPa PGS after a 72-h drug discontinuation at either a higher (U0126 [30 μM] and MK2206 [9 μM]) or lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined dose ( Fig. 6 ). No significant differences in cell proliferation of either EES (-P, -S, or -M) or NEES (-P, -S, or -M) (See  Supplementary Fig. S5 ) grown on substrates of varying stiffness (2- or 30-kPa PGS, or plastic) were observed after a 72-h drug discontinuation at either a higher (U0126 [30 μM] and MK2206 [9 μM]) or lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined dose.\nCell proliferation after a 72-h drug discontinuation was significantly increased compared to that after a 48-h treatment in DES, EES (EES-P, EES-S and EES-M) and NEES (NEES-P, NEES-S and NEES-M) grown on substrates of varying stiffness (2- or 30-kPa PGS, or plastic) at a lower combined dose (See  Supplementary Fig. S6 ). At a higher combined dose, cell proliferation after a 72-h drug discontinuation was significantly increased in DES grown on rigid substrates (30-kPa PGS or plastic), and EES-M grown on substrates of varying stiffness (2- or 30-kPa PGS, or plastic) (See  Supplementary Fig. S6 ).\nLC3-positive puncta, a marker for autophagy, were observed in DES, EES (-P, -S, and-M), and NEES (-P, -S, and-M) treated with MK2206 ( Fig. 7A ). Cell proliferation of DES after a 72-h discontinuation of combination U0126 (30 μM) and MK2206 (9 μM) with chloroquine (100 μM) was significantly lower than that without chloroquine when DES were grown on rigid substrates (30-kPa PGS or plastic) ( Fig. 7B ).\n\nThe present study showed a synergic effect of combined treatment with U0126 and MK2206 on DES, whereas an additive or antagonistic effect was observed on EES. The present study supports our speculation that cotargeting the PI3K/AKT/mTOR and RAF/MEK/ERK pathways may be effective for treatment of endometriosis 2 .\nIn the present study, we observed that the inhibition of cell proliferation was significantly higher in DES grown on plastic than those grown on 2- or 30-kPa PGS. One study evaluated a total of 18 small-molecule known or suspected inhibitors of cell proliferation in lung fibroblasts grown on soft (1-kPa PGS) or rigid (glass) substrates 5 . The study investigators identified compounds with both increased and decreased potency on soft relative to rigid substrates, in addition to those with equivalent efficacy irrespective of substrate stiffness 5 . These findings and our present findings suggest that when drug screening assays are performed in rigid plastic/glass, drug efficacy may be under- or over-estimated.\nAkt is well known for its anti-apoptotic activity 9 . However, the level of apoptosis as evaluated by Annexin V-positive cells did not appear to be sufficiently explained for the inhibition of cell proliferation in cells treated with MK2206 alone. Studies showed that Akt knockdown or inactivation with small-molecule inhibitors did not induce significant apoptosis 10 11 . Another mechanism may be responsible for inhibition of cell proliferation by MK2206. Our previous western blot analysis showed that MK2206 alone significantly increased levels of phosphorylated ERK in DES compared to those of vehicle-treated cells 2 . A hyperactivated ERK-driven transcriptional induction of the cyclin-dependent kinase inhibitor p21 and cyclin D1 triggers a massive accumulation of both cyclin D1 and p21, leading to cell cycle arrest by p21 12 . p21 mediates the tumor suppressor p53-dependent G1 growth arres 13 . When the cell cycle is blocked, while growth-promoting pathways remain active, cells continue to grow in size and undergo cellular senescence 12 . Cyclin D1 is the driving force of cell cycle transition from G1 to S phase in proliferating cells 14 . However, paradoxically, senescent cells have much higher levels of cyclin D1 than proliferating cells 15 16 . The present study showed the presence of beta-galactosidase activity, and increased cyclin D1 and p21 mRNA levels, which are biomarkers for cellular senescence 17 , in MK2206-treated cells. We speculated that abnormal hyperactivation of ERK through MK2206 may promote cellular senescence and result in inhibition of proliferation of MK2206-treated cells.\nThe present results showed that cell proliferation after discontinuation of the combined treatment was significantly higher in DES grown on 30-kPa PGS or plastic than those grown on 2-kPa PGS. However, we observed no significant effects of varying stiffness (2- or 30-kPa PGS, or plastic) on cell proliferation of either EES or NEES after drug discontinuation. These findings suggest that DES grown on a rigid substrate may have more potential to relapse than those grown on a soft substrate. Our findings appear to agree with the clinical evidence of a high recurrence rate following medical treatment in endometriosis 18 . It may be necessary to interrupt mechanical interactions between endometriotic cells and their surrounding ECM to prevent recurrence after medical treatment. The present  in vitro  findings may not support the future clinical use of the combined treatment with U0126 and MK2206 in patients with DIE, because of high cell survival and proliferation after drug discontinuation.\nIn the present study, we further attempted to investigate a potential mechanism underlying cell survival in DES treated with combination U0126 and MK2206. We observed a significantly higher percentage of Annexin V-positive cells in DES than in EES-P, -S, and -M when treated with combination U0126 and MK2206. Nevertheless, we observed higher proliferation of DES after drug discontinuation than of EES-P and EES-S when cells were grown on rigid substrates (30-kPa PGS or plastic). Studies showed that Akt knockdown or inactivation with small-molecule inhibitors markedly increased autophagy 17 19 20 21 . Autophagy is a highly conserved process in eukaryotes in which organelles, proteins, or lipids are sequestered into double-membrane vesicles termed autophagosomes for degradation and eventual recycling 22 . Inhibiting autophagy can either promote or inhibit cell death depending on the conditions and agents used 23 . Previous studies showed that MK2206 treatment induced autophagy in various cells types, and suppression of autophagy enhances cell death in an intracranial glioma mouse model 19  and in melanoma cells 21 , whereas it inhibits cell death in PTEN-mutant gastric cancer cells 20 . A recent study demonstrated upregulation of autophagy in ovarian endometriosis 24 . In addition, a recent study showed that hydroxychloroquine, an autophagy inhibitor, could decrease lesion numbers and disrupt lesion histopathology in a mouse model of endometriosis 25 . The present histochemical analysis revealed the presence of LC3-positive puncta in MK2206-treated DES 26 . In addition, we observed significantly lower proliferation of DES after discontinuation of treatment with U0126, MK2206, and chloroquine than with U0126 and MK2206 when cells were grown on rigid substrates. The present findings suggest that MK2206 treatment may induce autophagy, which may inhibit cell death, resulting in cell survival from combined treatment with U0126 and MK2206 and subsequent cell proliferation. However, the present analysis has limitations. The appearance of LC3-positive puncta does not necessarily indicate high levels of active autophagy 26 . In addition, most currently available chemical inhibitors of autophagy, including chloroquine, hydroxychloroquine, and bafilomycin A1, are not entirely specific 27 . Further studies are required to determine whether autophagy is involved in the high relapse rate of endometriosis after medical treatment.\nAnother potential explanation is that stem-like cells in endometriosis and menstrual endometrium of patients with endometriosis may be responsible for high cell survival and proliferation after discontinuation of combined treatment with U0126 and MK2206. A growing body of evidence suggests that endometriosis may arise from stem cells 28 . It has been proposed that endometrial stem/progenitor cells with associated niche cells are abnormally shed during menses, which may then implant into the peritoneal cavity by retrograde menstruation 28 . Endometriosis is a benign disease. However, studies have shown that endometriosis shares many aspects with cancer. It has been proposed that small subsets of cancer cells with extremely high tumorigenic potential, termed cancer stem cells (CSCs) or stem-like cancer cells, are responsible for relapse after cancer treatments such as chemotherapy or radiotherapy 29 30 . A recent study demonstrated that inhibition of cancer stemness effectively suppressed relapse and metastasis in a pancreatic cancer xenograft model 31 . In addition, preclinical data suggest that autophagy plays a crucial role in the origin, maintenance, and systemic distribution of CSCs 32 . Recent studies showed that pharmacologically altering CSC-related autophagy can overcome CSC resistance 33 34 . These findings led us to speculate that autophagy in stem-like cells in endometriosis may play a role in recurrence after medical treatment. Further studies are required to characterize DES and EES-M that can survive combined treatment with U0126 and MK2206 and subsequently proliferate after discontinuation of the combined treatment. Such investigations would provide further information for developing target therapies that prevent or minimize recurrence after medical treatment for endometriosis.\nThe present  in vitro  model has many limitations: endometriotic tissue and endometrium are composed of multiple cell types and extracellular matrix, but in the present study, only endometriotic and endometrial stromal cells were cultured based on our previous findings. Second, endometriotic tissue and endometrium are three-dimensional (3D), but the present studies used a conventional two-dimensional (2D) culture system. 3D  in vitro  models have been considered to span the gap between 2D cell cultures and whole-animal systems 35 36 . Further efforts are required to develop better culture systems that mimic the cellular complexity typical of  in vivo  endometriotic tissues.\nIn conclusion, the present study showed that combined treatment with U0126 and MK2206 synergistically inhibited cell proliferation of DES. However, cell proliferation of DES after drug discontinuation was higher than that of EES-P and EES-S when cells were grown on rigid substrates. The present  in vitro  findings may not support the future clinical use of the combined treatment with U0126 and MK2206 in patients with DIE. Further studies are required to investigate the mechanisms underlying high cell survival and proliferation after drug discontinuation for developing target therapies that prevent recurrence.\n\nPatients aged 20–37 years undergoing laparoscopy for endometriosis were recruited at CHU Clermont-Ferrand, France. None of the women had received hormonal therapy and none used intrauterine contraception for at least 6 months prior to surgery. Recruited patients had regular menstrual cycles (26–32 days) with confirmation of their menstrual history. Endometrial and endometriotic samples from 73 patients who had histological evidence of rectovaginal DIE were used for the present analysis. In addition, endometrial tissues from 21 patients without endometriosis were obtained. The clinical characteristics of patients are shown in  Supplementary Table S2 . The research protocol was approved by the Consultative Committee for Protection of Persons in Biomedical Research (CPP) of the Auvergne (France) region. All experiments were performed in accordance with the approved guidelines and regulations. Informed written consent was obtained from each patient prior to tissue collection.\nDES, EES, and NEES were isolated as previously described ( Supplementary methods ) 2 8 37 38 39 . Cells at passage 1 were used for experiments. The numbers of samples of DES, EES, and/or NEES used for each experiment are summarized in  Supplementary Table S3 . Immunofluorescence staining was performed to determine the purity of the isolated EES, NEES and DES as previously described 2 8 37 38 39 .\nStiffness-controlled 96-well plates were prepared using modifications to the protocol of Syed  et al . 40  (See  Supplementary Methods ).\nCell proliferation assays were performed using the CellTiter 96 ®  AQueous One Solution Cell Proliferation Assay (MTS) (Promega, Charbonnières-les-Bains, France), as previously described 37 38 39 . Briefly, cells (5 × 10 3  cells per well) from the same samples were plated on 2- or 30-kPa PGS or plastic in triplicate in 96-well plates. After 2 h at 37 °C and 5% CO 2  to allow cell adhesion and spreading, drugs were added at the indicated concentration with 100 μL culture media (2% charcoal-stripped FBS), individually or in combinations. U0126 (Selleck Chemicals, Houston, TX, USA) or MK2206 (Selleck Chemicals) were dissolved in dimethyl sulfoxide (DMSO) (Life Technologies). Chloroquine (Sigma-Aldrich) was dissolved in phenol red-free DMEM/F-12. The Chou-Talalay model calls for cytotoxic agents to be used at a fixed dose ratio 41 , so we elected to use U0126 and MK2206 in a 10:3 molar ratio based on the results of our previous study 2 . To calculate the combination index (CI) after 48 h of treatment, we used five different doses of U0126 and MK2206. To evaluate the effects of the combination of U0126 and MK2206 on inhibition of cell proliferation and cell survival after drug discontinuation, cells from the same samples were divided into two: one set was used to evaluate inhibition of cell proliferation after a 48-h treatment and the other set was used to evaluate cell proliferation of viable cells 72 h after drug discontinuation. We used two different doses of U0126 and MK2206 based on the results of prior experiments for CI. To evaluate inhibition of cell proliferation after the 48-h treatments, 20 μL of MTS were added to all wells and incubated for 2 h at 37 °C. To evaluate cell proliferation of viable cells after the 72-h drug discontinuations, cells were washed twice with PBS after a 48-h treatment, followed by a 72-h culture in drug-free medium with 10% FBS. Then, 20 μL of MTS were added to all wells and incubated for 2 h at 37 °C. Prior to absorbance measurements, 80 μL of the MTS:medium solution were transferred from each well into a well of a new 96-well plate to avoid background absorbance from the gels. Absorbance in the no-gel 96-well plate was measured at 490 nm (Spectra Max Plus, Molecular Devices, Sunnyvale, CA, USA). Percent cell proliferation was calculated as percent of vehicle control. CalcuSyn software (Biosoft, Great Shelford, Cambridge, UK) was used to calculate the CI according to the median-effect method of Chou and Talalay 42 43 . CI values <0.9, 0.9–1.1, and >1.1 represent synergism, additivity, and antagonism, respectively.\nCells (1 × 10 6  cells) were seeded onto Primaria flasks (BD Biosciences). After 2 h at 37 °C and 5% CO 2  to allow for cell adhesion and spreading, cells were incubated with culture media (2% charcoal-stripped FBS) containing either U0126 alone (30 μM) (Sigma-Aldrich), MK2206 alone (9 μM) (Sigma-Aldrich), a combination of U0126 (30 μM) and MK2206 (9 μM), or vehicle (DMSO) for 24 h. Cells were stained with Annexin V-FITC and PI (Annexin V kit, Beckman Coulter, Villepinte, France) and evaluated for apoptosis by flow cytometry analyses using a BD LSRII flow cytometer (BD Biosciences) according to the manufacturer’s protocol. Both early apoptotic (Annexin V-positive, PI-negative) and late (Annexin V-positive and PI-positive) apoptotic cells were included in cell death determinations.\nImmunofluorescence staining for LC3A/B (D2H10, 1:100, Cell Signaling, Danvers, MA, USA) was performed. Fluorescence histochemical detection of SA-βgal activity was performed according to the protocol published by Debacq-Chainiaux  et al . 44 . (See  Supplementary Methods ).\nCells were seeded onto 24-well plates (5 × 10 4  cells per well). After 2 h at 37 °C and 5% CO 2  to allow for cell adhesion and spreading, cells were incubated with culture media (2% charcoal-stripped FBS) containing either U0126 (30 μM) (Sigma-Aldrich), MK2206 (9 μM) (Sigma-Aldrich), U0126 (30 μM) and MK2206 (9 μM), or vehicle (DMSO) only for 24 h. Total RNA was extracted using the Qiagen RNeasy Mini Kit according to the manufacturer’s instructions (Qiagen, Courtaboef, France) as previously described 2 8 37 38 39 . RNA yield and integrity were analyzed using the RNA 6000 Pico kit and the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) as previously described 2 8 37 38 39 . mRNA levels of cyclin D1, p53, and p21 WAF1/Cip1  (p21) were measured by quantitative real-time RT-PCR with a Light Cycler (Roche, Mannheim, Germany) as previously described 2 8 37 38 39 . Here, Primer sets are shown in  Supplementary Table S4 .\nThe STATA program version 12 (StataCorp, College Station, TX, USA) was used for statistical analysis. Comparisons between different groups were made using one-way analysis of variance following Scheffé’s method, the Mann-Whitney  U  test, or the Wilcoxon matched pairs signed-ranks test. Statistical significance was defined as p < 0.05.\n\nHow to cite this article : Matsuzaki, S.  et al . Effects of U0126 and MK2206 on cell growth and re-growth of endometriotic stromal cells grown on substrates of varying stiffness.  Sci. Rep. \n 7 , 42939; doi: 10.1038/srep42939 (2017).\nPublisher's note:  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.","source_license":"CC0","license_restricted":false}