u-0126 and Endometriosis

Topics: Animals; Butadienes; Cell Line; Cell Proliferation; Cell Survival; Chromones; Cytokines; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Endometriosis; Epigenesis, Genetic; Female; Gene Expression Regulation; Humans; MAP Kinase Signaling System; Mice; Morpholines; Nitriles; Proto-Oncogene Proteins c-akt; Receptors, Progesterone

Endometriosis is an estrogen-dependent and progesterone-resistant gynecological inflammatory disease of reproductive-age women. Current hormonal therapies targeting estrogen can be prescribed only for a short time. It indicates a need for non-hormonal therapy. ERK1/2 and AKT pathways control several intracellular signaling molecules that control growth and survival of cells. Objectives of the present study are to determine the dual inhibitory effects of ERK1/2 and AKT pathways: (i) on proliferation, survival, and apoptosis of human endometrioitc epithelial cells and stromal cells in vitro; (ii) on growth and survival of endometrioitc lesions in vivo in xenograft mouse model of endometriosis of human origin; and (iii) establish the associated ERK1/2 and AKT downstream intracellular signaling modules in the pathogenesis of endometriosis. Our results indicated that combined inhibition of ERK1/2 and AKT pathways highly decreased the growth and survival of human endometriotic epithelial cells and stromal cells in vitro and suppressed the growth of endometriotic lesions in vivo compared to inhibition of either ERK1/2 or AKT pathway individually. This cause-effect is associated with dysregulated intracellular signaling modules associated with cell cycle, cell survival, and cell apoptosis pathways. Collectively, our results indicate that dual inhibition of ERK1/2 and AKT pathways could emerge as potential non-hormonal therapy for the treatment of endometriosis.

Topics: Animals; Butadienes; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromones; Drug Therapy, Combination; Endometriosis; Female; Humans; MAP Kinase Signaling System; Mice; Morpholines; Nitriles; Proto-Oncogene Proteins c-akt; Xenograft Model Antitumor Assays

We aimed at exploring the positive feedback loop in eutopic and ectopic endometrial glandular epithelial cells (EuECs and EECs) in endometriosis.. Normal epithelial cells (NECs), EuECs and EECs were treated with fibroblast growth factor (FGF)2, FGF2 neutralizing antibody, mitogen-activated protein kinases (MAPKs) inhibitors U0126 and PD98059. FGF2 protein level was detected by enzyme-linked immunosorbent assay (ELISA). The expressions of FGF2, FGF receptor 1 (FGFR1), extracellular signal-regulated kinase (ERK)1/2/pERK1/2 and Sproutys (SPRYs) (Sprouty1, Sprouty2, Sprouty4) and dual specificity phosphatase 6 (DUSP6) were detected by Western blot. The mRNA levels of FGF2, FGFR1 (FGF receptor 1), SPRYs (Sprouty1, Sprouty2, Sprouty4) and DUSP6 mRNA were detected by RT-PCR.. Among treatment groups, the content of FGF2 in EuECs and EECs was significantly higher than that in NECs (p < 0.05). The mRNA and protein levels of FGF2, FGFR1, SPRYs (Sprouty1, Sprouty2, Sprouty4) and DUSP6 in EuECs and EECs were increased after adding FGF2 (p < 0.05), but decreased after adding FGF2 neutralizing antibody, no significant change was found in NECs (p > 0.05). The inhibitory effect of PD9805 on NECs was not significantly different from that of U0126 (p > 0.05); however, the inhibitory effects of PD9805 on EuECs and EECs were significantly lower than those of U0126 (p< 0.05).. The positive feedback loop existed in EuECs and EECs, but maybe not in NECs. The results may provide the guideline to treat endometriosis patients.

Topics: Antibodies, Neutralizing; Butadienes; Cell Line; Dual Specificity Phosphatase 6; Endometriosis; Endometrium; Epithelial Cells; Female; Fibroblast Growth Factor 2; Flavonoids; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nerve Tissue Proteins; Nitriles; Phosphoproteins; Signal Transduction

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

To examine the involvement of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) in the induction of interleukin-6 (IL-6) by tumor necrosis factor-alpha (TNF-alpha) in endometriotic stromal cells (ESC).. Prospective study.. Department of Obstetrics and Gynecology, Tottori University Hospital, Yonago, Japan.. Twelve patients who underwent laparoscopic surgery.. Endometriotic stromal cells were obtained from chocolate cyst linings of the ovary.. We determined the effect of TNF-alpha on the production of IL-6 and the effect of inhibitors for NF-kappaB and the MAPK pathway on IL-6 production using ELISA. Western blottings and electrophoretic mobility shift assays were used to detect activation of NF-kappaB and extracellular signal-regulated kinase 1/2 (ERK1/2).. The addition of TNF-alpha (0.1 ng/mL) significantly increased IL-6 protein in endometriotic stromal cells. Western blottings and electrophoretic mobility shift assays revealed that incubation with TNF-alpha induced degradation of inhibitor kappaB (I kappaB) and expression of phosphorylated ERK1/2. The NF-kappaB inhibitor (TPCK) and MAPK inhibitor (U0126) blocked the TNF-alpha-induced IL-6 expression. Electrophoretic mobility shift assay revealed that U0126 attenuated activator protein-1 (AP-1) activation induced by TNF-alpha.. These findings demonstrate that NF-kappaB and AP-1 activation is critical for TNF-alpha-induced IL-6 expression in endometriotic stromal cells. Novel therapeutic modalities targeting these molecules may be possible in the near future.

Topics: Blotting, Western; Butadienes; Electrophoresis; Endometriosis; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Female; Humans; I-kappa B Proteins; Interleukin-6; Mitogen-Activated Protein Kinases; NF-kappa B; Nitriles; Phosphorylation; Prospective Studies; Stromal Cells; Transcription Factor AP-1; Tumor Necrosis Factor-alpha