mk-2206 and Endometriosis

Fibrinogen alpha chain (FGA), a cell adhesion molecule, contains two arginyl-glycyl-aspartic acid (RGD) cell adhesion sequences. Our previous study demonstrated that FGA, as an up-regulated protein in endometriosis (EM), was closely related to disease severity and involved in the development of EM. However, the biological functions and underlying mechanism of FGA in EM have not been fully understood. To explore the roles of FGA in EM, we analyzed the effects of FGA on the biological behaviors of human primary eutopic endometrial stromal cells (EuESC). The results indicated FGA knockdown suppressed the migration and invasion ability of EuESC, which also altered the distribution of cytoskeletal filamentous and cell morphology. Western blot analysis demonstrated that knockdown of FGA attenuated the migration-related protein levels of vimentin and matrix metallopeptidase 2 (MMP-2), but not integrin subunit alpha V (ITGAV) and integrin subunit beta 3 (ITGB3). Meanwhile, integrin-linked transduction pathways were detected. We found FGA knockdown significantly suppressed the expression of focal adhesion kinase (FAK) level and protein kinase B (AKT) phosphorylation, without extracellular-signal-regulated kinase (ERK) dependent pathways. Treatment with the AKT inhibitor MK2206 or RGD antagonist highly decreased the effects of FGA on the migration and invasion of EuESC. RGD antagonist treatment strongly inhibited FAK- and AKT-dependent pathways, but not ERK pathways. Our data indicated that FGA may enhance the migration and invasion of EuESC through RGD sequences binding integrin and activating the FAK/AKT/MMP-2 signaling pathway. This novel finding suggests that FGA may provide a novel potential approach to the treatment of EM, which provides a new way to understand the pathogenesis of EM.

Topics: Adult; Cell Movement; Endometriosis; Endometrium; Female; Fibrinogen; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation; Heterocyclic Compounds, 3-Ring; Humans; Matrix Metalloproteinase 2; Oligopeptides; Proto-Oncogene Proteins c-akt; Stromal Cells; Wound Healing

A high recurrence rate after medical treatment is a major clinical problem for patients with endometriosis. Here, we have evaluated the in vitro effects of combined treatment with MK2206 (an AKT inhibitor) + chloroquine on cell growth and regrowth of endometriotic stromal cells and the in vivo effects on endometriotic implants in a mouse xenograft model of endometriosis.. We evaluated the effects of autophagy inhibition by knockdown of the ATG13, Beclin-1 and ATG12 genes and pharmacological agents (chloroquine, bafilomycin A1 or 3-methyalanine) individually and in combination with MK2206 on cell growth and/or cell regrowth of endometriotic stromal cells in vitro. Furthermore, we evaluated treatment with MK2206 + chloroquine on endometriotic implants in a mouse xenograft model of endometriosis.. Combined treatment with MK2206 and chloroquine markedly reduced cell growth and regrowth after discontinuation of treatment in endometriotic stromal cells compared with cells treated with either drug alone. Autophagy inhibition by ATG13, Beclin-1 or ATG12 gene knockdown only affected regrowth of endometriotic stromal cells, but not endometrial stromal cells from the same patients, after a 72 h discontinuation of the combined treatment. Furthermore, combined treatment reduced the size of endometriotic implants, whereas no effects on endometriotic implants treated with either drug alone were observed in a mouse xenograft model of endometriosis.. The present findings suggest that a novel strategy for treatment of endometriosis may involve decreasing the number of endometriotic cells that can survive treatment and then preventing regrowth by autophagy inhibition.

Topics: Adult; Animals; Autophagy; Cell Proliferation; Cells, Cultured; Chloroquine; Disease Models, Animal; Drug Therapy, Combination; Endometriosis; Female; Heterocyclic Compounds, 3-Ring; Humans; Mice; Young Adult

Excess fibrosis may lead to chronic pain, scarring, and infertility as endometriosis develops and progresses. The pathogenesis of endometriosis has been linked to transforming growth factor-β (TGF-β), the most potent promoter of fibrosis.. Levels of NR4A1 and P-NR4A1 protein in human endometrial and endometriotic tissue were assessed by western blotting and immunohistochemistry. The expression levels of fibrotic markers in stromal cells were evaluated by real-time PCR. The degree of fibrosis in mouse endometriotic lesions was detected by Masson trichrome and Sirius red staining.. The level of phosphorylated-NR4A1 was higher in ovarian endometriotic tissue than in normal endometrium, and long-term TGF-β1 stimulation phosphorylated NR4A1 in an AKT-dependent manner and then promoted the expression of fibrotic markers. Furthermore, inhibition of NR4A1 in stromal cells increased the TGF-β1-dependent elevated expression of fibrotic markers, and loss of NR4A1 stimulated fibrogenesis in mice with endometriosis. Additionally, Cytosporone B (Csn-B), an NR4A1 agonist, effectively decreased the TGF-β1-dependent elevated expression of fibrotic markers in vitro and significantly inhibited fibrogenesis in vivo.. NR4A1 can regulate fibrosis in endometriosis and may serve as a new target for the treatment of endometriosis.

Topics: Adult; Animals; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Connective Tissue Growth Factor; Disease Models, Animal; Endometriosis; Endometrium; Female; Fibronectins; Fibrosis; Heterocyclic Compounds, 3-Ring; Humans; Mice; Mice, Nude; Microscopy, Fluorescence; Nuclear Receptor Subfamily 4, Group A, Member 1; Phenylacetates; Phosphorylation; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Stromal Cells; Transforming Growth Factor beta; Transplantation, Heterologous; Up-Regulation; Young Adult

Topics: Autophagy; Chloroquine; Endometriosis; Female; Heterocyclic Compounds, 3-Ring; Humans

Topics: Autophagy; Chloroquine; Endometriosis; Female; Heterocyclic Compounds, 3-Ring; Humans; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases

Endometriosis is a common gynecological disorder responsible for infertility and pelvic pain. A complete cure for patients with endometriosis awaits new targets and strategies. Here we show that U0126 (a MEK inhibitor) and MK2206 (an AKT inhibitor) synergistically inhibit cell growth of deep endometriotic stromal cells (DES) grown on polyacrylamide gel substrates (PGS) of varying stiffness (2 or 30 kilopascal [kPa]) or plastic in vitro. No significant differences in cell proliferation were observed among DES, endometrial stromal cells of patients with endometriosis (EES) from the proliferative phase (P), EES-S (secretory phase) and EES-M (menstrual phase) compared to cells grown on a substrate of the same stiffness at both higher (U0126 [30 μM] and MK2206 [9 μM]) and lower (U0126 [15 μM] and MK2206 [4.5 μM]) combined doses. However, cell re-growth of DES after drug discontinuation was higher than that of EES-P and EES-S when cells were grown on rigid substrates at both combined doses. Combination U0126 and MK2206 treatment is more effective than each drug alone in cell growth inhibition of DES. However, further studies are required to investigate the mechanisms underlying high cell survival and proliferation after drug discontinuation for developing target therapies that prevent recurrence.

Topics: Acrylic Resins; Adult; Apoptosis; Butadienes; Cell Proliferation; Cells, Cultured; Cellular Senescence; Drug Combinations; Endometriosis; Endometrium; Female; Hardness; Heterocyclic Compounds, 3-Ring; Humans; Nitriles; Plastics; Stromal Cells; Young Adult

The pathogenesis of endometriosis remains unclear, and relatively little is known about the mechanisms that promote establishment and survival of the disease. Previously, we demonstrated that v-akt murine thymoma viral oncogene homolog (AKT) activity was increased in endometriosis tissues and cells from ovarian endometriomas and that this increase promoted cell survival as well as decreased levels of progesterone receptor. The objective of this study was to demonstrate a role for AKT in the establishment of ectopic lesions. First, a dose-dependent inhibition of AKT in stromal cells from human ovarian endometriomas (OSIS) as well as endometrial stromal cells from disease-free patients (ESC) with the allosteric AKT inhibitor MK-2206 was demonstrated by decreased levels of phosphorylated (p)(Ser473)-AKT. Levels of the AKT target protein, p(Ser256)-forkhead box O1 were increased in OSIS cells, which decreased with MK-2206 treatment, whereas levels of p(Ser9)-glycogen synthase kinase 3β did not change in response to MK-2206. Although MK-2206 decreased viability of both OSIS and ESC in a dose-dependent manner, proliferation of OSIS cells was differentially decreased significantly compared with ESC. Next, the role of hyperactive AKT in the establishment of ectopic lesions was studied using the bigenic, PR(cre/+)Pten(f/+) heterozygous mouse. Autologous implantation of uterine tissues was performed in these mice. After 4 weeks, an average of 4 ± 0.33 lesions per Pten(f/+) mouse and 7.5 ± 0.43 lesions in the PR(cre/+)Pten(f/+) mouse were found. Histological examination of the lesions showed endometrial tissue-like morphology, which was similar in both the Pten(f/+) and PR(cre/+)Pten(f/+) mice. Treatment of mice with MK-2206 resulted in a significantly decreased number of lesions established. Immunohistochemical staining of ectopic lesions revealed decreased p(Ser473)-AKT and the proliferation marker Ki67 from MK-2206-treated mice compared with vehicle-treated mice. Furthermore, levels of FOXO1 and progesterone receptor increased in lesions of mice receiving MK-2206. These results demonstrate that heightened AKT activity plays an active role in the establishment of ectopic endometrial tissues.

Topics: Animals; Cell Proliferation; Cell Survival; Cells, Cultured; Crosses, Genetic; Disease Models, Animal; Endometriosis; Endometrium; Female; Forkhead Box Protein O1; Forkhead Transcription Factors; Heterocyclic Compounds, 3-Ring; Humans; Mice; Mice, Transgenic; Ovarian Diseases; Ovary; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Signal Transduction; Stromal Cells