u-0126 and Endometrial-Neoplasms

Endometrial cancer is the most common, and second most lethal, gynecological malignancy, and its rates of incidence and death are growing. This is likely attributable to increased numbers of high-risk type II endometrial cancers which account for ~30% of cases but ~75% of deaths due to their aggressive and metastatic behaviour. Histopathological and in vitro functional studies suggest that aberrant TGFβ1 signaling may contribute to endometrial cancer development and the acquisition of invasive/metastatic characteristics. However, little is known about the cellular and molecular mechanisms of TGFβ1 in high-risk endometrial cancers. In the present study, we examined the roles and mechanisms of TGFβ1 on cell adhesion and motility in type II endometrial cancer cell lines, KLE and HEC-1B. We show that treatment with TGFβ1 increases cell adhesion to vitronectin and transwell cell migration. We also demonstrate that TGFβ1 treatment increases integrin β3 and αv mRNA and protein levels via SMAD-independent MEK-ERK1/2 signaling. Importantly, siRNA depletion or antibody-mediated blocking of integrin αvβ3 reversed the effects of TGFβ1 on cell adhesion and migration. Our results suggest that TGFβ1-MEK-ERK1/2-integrin αvβ3 signaling could contribute to the invasive behaviour of high-risk endometrial cancer by promoting cell adhesion and migration.

Topics: Butadienes; Cell Adhesion; Cell Line, Tumor; Cell Movement; Endometrial Neoplasms; Female; Humans; Integrin alphaVbeta3; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1; Up-Regulation

Radiation therapy is a key therapeutic strategy for endometrial carcinomas. However, biomarkers that predict radiosensitivity and drugs to enhance this sensitivity have not yet been established. We aimed to investigate the roles of TP53 and MAPK/PI3K pathways in endometrial carcinomas and to identify appropriate radiosensitizing therapeutics. D10 values (the irradiating dose required to reduce a cell population by 90%) were determined in eight endometrial cancer cell lines with known mutational statuses for TP53, PIK3CA, and KRAS. Cells were exposed to ionizing radiation (2-6Gy) and either a dual PI3K/mTOR inhibitor (NVP-BEZ235) or a MEK inhibitor (UO126), and their radiosensitizing effects were evaluated using clonogenic assays. The effects of silencing hypoxia-inducible factor-1 α (HIF-1α) expression with small interfering RNAs (siRNAs) were evaluated following exposure to ionizing radiation (2-3Gy). D10 values ranged from 2.0 to 3.1Gy in three cell lines expressing wild-type TP53 or from 3.3 to more than 6.0Gy in five cell lines expressing mutant TP53. NVP-BEZ235, but not UO126, significantly improved radiosensitivity through the suppression of HIF-1α/vascular endothelial growth factor-A expression. HIF-1α silencing significantly increased the induction of the sub-G1 population by ionizing radiation. Our study data suggest that TP53 mutation and PI3K pathway activation enhances radioresistance in endometrial carcinomas and that targeting the PI3K/mTOR or HIF-1α pathways could improve radiosensitivity.

Topics: Butadienes; Carcinoma, Endometrioid; Cell Line, Tumor; Endometrial Neoplasms; Enzyme Inhibitors; Female; Genes, p53; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Radiation Tolerance; TOR Serine-Threonine Kinases; Vascular Endothelial Growth Factor A

Autocrine motility factor (AMF) as a cytokine and a growth factor, is known to regulate tumor cell growth and motility in the progress of various human malignant tumors, however, its role in endometrial cancer (EC) has not been fully studied. In the present study, using immunohistochemistry, we found that AMF was highly expressed in EC tissues compared with normal endometrial tissues and tissue micrioarray technology showed positive correlation between AMF expression and epithelial-to-mesenchymal transition (EMT) related markers E-cadherin, vimentin and Snail. Next, we detected that silencing of AMF by stable transfection with shRNA induced mesenchymal-to-epithelial transition phenotype in Ishikawa and HEC-1B cells by qRT-PCR, western blotting and immunofluorescence. Gene expression profile revealed that AMF silencing resulted in altered expression of EMT related molecular mediators including Snail and transforming growth factor β receptor 1, and involvement of mitogen-activated protein kinase (MAPK) signaling pathway. Additionally, we found that EMT related markers were downregulated with pretreatment of the MAPK-specific inhibitor U0126 by western blotting. The present study is the first to support a role for AMF mediating EMT in endometrial cancer through MAPK signaling. Therefore, AMF may provide a potential prognostic and therapeutic target in preventing EC progression.

Topics: Biomarkers, Tumor; Butadienes; Cell Line, Tumor; Endometrial Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Nitriles; Receptors, Autocrine Motility Factor

Osteopontin (OPN) is an Extracellular Matrix (ECM) molecule and is involved in many physiologic and pathologic processes, including cell adhesion, angiogenesis and tumor metastasis. OPN is a well-known multifunctional factor involved in various aspects of cancer progression, including endometrial cancer. In this study, we examined the significance of OPN in endometrial cancer.. The proliferation, migration and invasion ability of HEC-1A cells were detected by Cell Counting Kit-8 (CCK-8), Wound scratch assay and transwell. Western blots were employed to detect the expression of Matrix metalloproteinase-2 (MMP-2) and epithelial-mesenchymal transition (EMT)-related factors in HEC-1A cells treated with rhOPN.. rhOPN promotes cell proliferation, migration and invasion in HEC-1A cells. rhOPN influenced EMT-related factors and MMP-2 expression in HEC-1A cells. rhOPN promoted HEC-1A cells migration, invasion and EMT through protein kinase B (PKB/AKT) and Extracellular regulated protein kinases (ERK1/2) signaling pathway.. These results may open up a novel therapeutic strategy for endometrial cancer: namely, rhOPN have important roles in controlling growth of endometrial of cancer cells and suggest a novel target pathway for treatment of this cancer.

Topics: Butadienes; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromones; Endometrial Neoplasms; Epithelial-Mesenchymal Transition; Female; Humans; Matrix Metalloproteinase 2; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; Nitriles; Osteopontin; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Signal Transduction; Vimentin

Given that prolonged exposure to estrogen and increased telomerase activity are associated with endometrial carcinogenesis, our objective was to evaluate the interaction between the MAPK pathway and estrogen induction of telomerase activity in endometrial cancer cells. Estradiol (E2) induced telomerase activity and hTERT mRNA expression in the estrogen receptor (ER)-α positive, Ishikawa endometrial cancer cell line. UO126, a highly selective inhibitor of MEK1/MEK2, inhibited telomerase activity and hTERT mRNA expression induced by E2. Similar results were also found after transfection with ERK 1/2-specific siRNA. Treatment with E2 resulted in rapid phosphorylation of p44/42 MAPK and increased MAPK activity which was abolished by UO126. The hTERT promoter contains two estrogen response elements (EREs), and luciferase assays demonstrate that these EREs are activated by E2. Exposure to UO126 or ERK 1/2-specific siRNA in combination with E2 counteracted the stimulatory effect of E2 on luciferase activity from these EREs. These findings suggest that E2-induction of telomerase activity is mediated via the MAPK pathway in human endometrial cancer cells.

Topics: Butadienes; Cell Line, Tumor; Endometrial Neoplasms; Enzyme Activation; Estradiol; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nitriles; Promoter Regions, Genetic; RNA Interference; RNA, Messenger; Signal Transduction; Telomerase

More than 25% of patients diagnosed with endometrial carcinoma have an invasive primary cancer accompanied by metastases. Gonadotropin-releasing hormone (GnRH) plays an important role in reproduction. In mammals, expression of GnRH-II is higher than GnRH-I in reproductive tissues. Here, we examined the effect of a GnRH-II agonist on the motility of endometrial cancer cells and its mechanism of action in endometrial cancer therapy.. Immunoblotting and immunohistochemistry (IHC) were used to determine the expression of the GnRH-I receptor protein in human endometrial cancer. The activity of MMP-2 in the conditioned medium was determined by gelatin zymography. Cell motility was assessed by invasion and migration assay. GnRH-I receptor si-RNA was applied to knockdown GnRH-I receptor.. The GnRH-I receptor was expressed in the endometrial cancer cells. The GnRH-II agonist promoted cell motility in a dose-dependent manner. The GnRH-II agonist induced the phosphorylation of ERK1/2 and JNK, and the phosphorylation was abolished by ERK1/2 inhibitor (U0126) and the JNK inhibitor (SP600125). Cell motility promoted by GnRH-II agonist was suppressed in cells that were pretreated with U0126 and SP600125. Moreover, U0126 and SP600125 abolished the GnRH-II agonist-induced activation of MMP-2. The inhibition of MMP-2 with MMP-2 inhibitor (OA-Hy) suppressed the increase in cell motility in response to the GnRH-II agonist. Enhanced cell motility mediated by GnRH-II agonist was also suppressed by the knockdown of the endogenous GnRH-I receptor using siRNA.. Our study indicates that GnRH-II agonist promoted cell motility of endometrial cancer cells through the GnRH-I receptor via the phosphorylation of ERK1/2 and JNK, and the subsequent, MAPK-dependent activation of MMP-2. Our findings represent a new concept regarding the mechanism of GnRH-II-induced cell motility in endometrial cancer cells and suggest the possibility of exploring GnRH-II as a potential therapeutic target for the treatment of human endometrial cancer.

Topics: Anthracenes; Butadienes; Carcinoma; Cell Line, Tumor; Cell Movement; Endometrial Neoplasms; Enzyme Activation; Female; Gene Knockdown Techniques; Gonadotropin-Releasing Hormone; Humans; Hydroxamic Acids; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Nitriles; Phosphorylation; Receptors, LHRH

Human potassium chloride cotransporter-1 (KCC1) gene is expressed in endometrial cancer and related to metastasis of endometrial cancer. However, whether KCC1 contributes to invasion and metastasis of endometrial cancer has not been thoroughly investigated. The purpose of this study is to research the alternation effect of insulin-like growth factor I (IGF-I) on the expression of KCC1 in endometrial cancer HEC-1B cells and to explore the mechanism of how KCC1 regulates the invasion ability of HEC-1B cells through the extracellular signal-regulated kinase (ERK) signaling pathway.. First, the inhibitive effect of RNA interference to KCC1 was detected by semiquantitative reverse transcriptase-polymerase chain reaction. Western blot was used to measure expression changes of KCC1 after exposure to IGF-I in the HEC-1B cells. The change in quantity of phosphorylated ERK1/2 (p-ERK1/2) and cell invasion ability also were measured. After RNA interference and treatment with U0126, the quantity of p-ERK1/2 and the cell invasion ability were measured again.. After the application of IGF-I on the HEC-1B cells, the expression of KCC1 and p-ERK1/2 increased dramatically, and the cell invasion ability advanced. RNA interference could inhibit the expression of KCC1, and the quantity of p-ERK1/2 and the cell invasion ability decreased even under the effect of IGF-I. Furthermore, after treatment with U0126, the cell invasion ability no longer advanced even under the effect of IGF-I either.. Insulin-like growth factors I can induce the upregulation of KCC1 gene, and KCC1 gene participates in the invasion ability of HEC-1B cells through the ERK signaling pathway.

Topics: Butadienes; Carcinoma; Cell Adhesion; Cell Line, Tumor; Cell Movement; Drug Evaluation, Preclinical; Endometrial Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; K Cl- Cotransporters; MAP Kinase Kinase 1; MAP Kinase Signaling System; Neoplasm Invasiveness; Nitriles; Protein Kinase Inhibitors; Symporters; Up-Regulation

Recently, a novel variant of ER-alpha, ER-alpha36 was identified and cloned. ER-alpha36 lacks intrinsic transcription activity and mainly mediates nongenomic estrogen signaling. Here, we studied the role of nongenomic estrogen signaling pathways mediated by ER-alpha36 in tamoxifen resistance and agonist action.. The cellular localization of ER-alpha36 was examined by immunofluorescence in MCF-7 cells and Hec1A cells. MCF-7 breast cancer cells, MCF-7 cells expressing recombinant ER-alpha36 (MCF-7/ER36), Hec1A endometrial cancer cells and Hec1A cells with siRNA knockdown of ER-alpha36 (Hec1A/RNAiER36) were treated with 17beta-estradial (E2) and tamoxifen (TAM) in the absence and presence of kinase inhibitor U0126 and LY294002. We examined phosphorylation of signaling molecules and the expression of c-Myc by immunoblotting, and tumor cell growth by MTT assay.. ER variant ER-alpha36 enhances TAM agonist activity through activation of the membrane-initiated signaling pathways in endometrial cancer, and that ER-alpha36 is involved in de novo and acquired TAM resistance in breast cancer.

Topics: Alternative Splicing; Antineoplastic Agents, Hormonal; Blotting, Western; Butadienes; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Chromones; Dose-Response Relationship, Drug; Endometrial Neoplasms; Enzyme Activation; Estrogen Receptor alpha; Estrogens; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Isoforms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; RNA Interference; Signal Transduction; Tamoxifen

The extracellular-regulated kinase (ERK) signaling pathway plays important roles in regulating the malignant potential of cancer cells in vitro. However, the effect of ERK signaling on the prognosis of human tumors is not clearly understood. The present study examined the expression of phosphorylated ERK1/2 (p-ERK1/2) as a hallmark of ERK activation, in relation to KRAS and BRAF mutations, in 63 endometrial cancer specimens with endometrioid-subtype, in order to clarify the prognostic value of p-ERK1/2 expression. Immmunohistochemical analysis revealed that 40 tumors (63%) expressed p-ERK1/2, with varying levels of expression. Total ERK1/2 expression was also evaluated in a subset of tumors; most cases expressed ERK1/2 constitutively but no correlation was observed with p-ERK expression, indicating that p-ERK1/2 staining was not due to ERK overexpression but to hyperactivation of ERK1/2. There was no statistically significant correlation between p-ERK1/2 expression and clinicopathological features, including patient age, International Federation of Gynecology and Obstetrics stage, pathological grade, myometrial invasion and lymph node metastasis. Sequencing analysis indicated that 23% of patients had a mutation in exon 1 of KRAS, whereas none of the patients had a mutation in exons 11 or 15 of BRAF, which are reportedly hot spots for mutation in many tumor types. There was no significant correlation between KRAS or BRAF status and p-ERK1/2 expression. Unexpectedly, patients with low p-ERK1/2 expression had significantly lower relapse-free survival (P = 0.041) and overall survival (P = 0.020). Multivariate Cox regression analysis indicated that p-ERK1/2 expression was an independent prognostic indicator for overall survival (P = 0.047). These findings suggest that ERK activation occurs in a KRAS- and BRAF-independent manner in endometrial cancer, and is associated with favorable prognosis.

Topics: Adult; Aged; Blotting, Western; Butadienes; Cell Line; Endometrial Neoplasms; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Nitriles; Phosphorylation; Prognosis; Proportional Hazards Models; Proto-Oncogene Proteins B-raf; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The fact that the genetic alterations of PTEN are frequently found in hormone-dependent cancers, such as endometrial, breast, and prostate cancers, might suggest the involvement of PTEN in the hormone-dependent cell growth of such tumors. Estrogen promotes the cell growth of the tumors by inducing peptide growth factors in part. We analyzed the possible involvement of PTEN in peptide-growth factor-dependent cell growth in endometrial carcinoma cells. PTEN-null Ishikawa cells were efficiently infected with recombinant adenovirus at 20 MOI (multiplicity of infection) to express PTEN protein. In PTEN-IK cells, phospho-Akt/PKB was down-regulated regardless of the consistent expression of Akt/PKB. The cell growth of parental IK cells was significantly stimulated by EGF and IGF-I, and PTEN-IK cells were further sensitized to the EGF-or IGF-I-growth stimulation. EGFR antibody could completely compromise the stimulatory effects of EGF in both cell lines. Wortmannin, a PI3K inhibitor, or UO126, a MAPK inhibitor, partly suppressed EGF-mediated cell growth stimulation in both cell lines. EGF augmented the level of phospho-Akt/PKB of PTEN-IK cells more effectively than that of parental IK cells. These results imply that the dysfunction of PTEN leads cells into a less-sensitive phenotype to peptide growth factors by constitutive activation of the PI3K/Akt/PKB signaling pathway in endometrial carcinoma.

Topics: Adenoviridae; Androstadienes; Antibodies; Blotting, Western; Butadienes; Cell Line; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA, Recombinant; Endometrial Neoplasms; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblast Growth Factor 2; Gene Expression; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor I; Mitogen-Activated Protein Kinases; Nitriles; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptor, ErbB-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Wortmannin

The extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway plays a critical role in the anticancer action in vitro. ERK1/2 activation or phosphorylation is responsible for increased cyclooxygenase-2 (COX-2) protein expression in some cancer cells treated with selective COX-2 inhibitor NS398. We determined the effect of NS398 on ERK signaling and the synergistic effect of combined treatment with NS398 and a specific MEK inhibitor U0126 on three human endometrial cancer cell lines: Ishikawa, HEC-1A and AN3CA cells. Results showed that NS398 and U0126 individually, and especially the combination of both exhibited profound anti-proliferation of all three cell lines in a time- and concentration-dependent manner by [3-(4, 5)-dimethylthiazol-z-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay. The phosphorylated ERK1/2 was up-regulated in HEC-1A and AN3CA cells, but the COX-2 protein expression was unchanged in the three cancer cell lines treated with NS398 alone. However, both phosphorylated ERK1/2 and COX-2 protein expression were concentration-dependently decreased in all three cell types by combined treatment with NS398 and U0126 assessed by western blot analysis. Simultaneously, the combination of NS398 and U0126 resulted in 2-fold increase in apoptosis of all three lines over that by the individual alone, and enhanced G0/G1 phase arrest of Ishikawa and HEC-1A cells induced by U0126 treatment determined by flow cytometry. The synergistic and complementary effects of combining NS398 and U0126 were found to be associated with activation of caspase-3, alterations of Bcl-2 family proteins and cell cycle regulatory proteins detected by western blot analysis. Taken together, these findings correlate with blocking MEK-ERK signaling cascade and down-regulating COX-2 protein expression in endometrial cancer cells with combination treatment of NS398 and U0126, suggesting that the combinatory use of NS398 and specific MEK inhibitors may be valuable for chemotherapy or chemoprevention of human endometrial cancer.

Topics: Blotting, Western; Butadienes; Cell Proliferation; Cyclooxygenase Inhibitors; Endometrial Neoplasms; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; MAP Kinase Kinase Kinases; Nitriles; Nitrobenzenes; Signal Transduction; Sulfonamides; Tumor Cells, Cultured

Fibroblast growth factors (FGFs) exert diverse effects resulting from their interaction with cognate receptors on target cells. Our current study was designed to examine the local production and action of two specific stromal-epithelial cell mediatory factors, keratinocyte growth factor (KGF) and FGF-10, in human endometrial carcinoma cells. The RT-PCR method was used to determine gene expression of KGF, FGF-10, and KGF receptor in human endometrial carcinoma cells (HEC-1) and human endometrial stromal cells. KGF mRNAs were expressed in both of these cell types. On the other hand, FGF-10 mRNA was detected only in the endometrial stromal cells, and KGF receptor mRNA was observed in the HEC-1 cells. The novel finding of the present study is that KGF is expressed in carcinoma cells and FGF-10 is expressed in human endometrial stromal cells. The distinct phosphorylation of ERK-1 and -2 (ERK1/2), which are members of the MAPK family, was observed when HEC-1 cells were treated with KGF or FGF-10. KGF and FGF-10 could induce the prompt phosphorylation of ERK1/2 and consequently stimulate DNA synthesis. KGF and FGF-10 did not activate the phosphorylation of Akt, protein kinase C, or signal transducer and activator of transcription-3. Blocking the MAPK pathway with the specific methyl ethyl ketone 1/2 inhibitor (U0126) completely neutralized the enhancement of cell proliferation induced by KGF and FGF-10. In addition, KGF and FGF-10 activated expressions of downstream nuclear transcription factors, such as Elk-1 and c-myc, but not c-fos. These results demonstrate for the first time that KGF and FGF-10 are capable of stimulating the growth of endometrial carcinoma cells via activating MAPK pathway through autocrine/paracrine fashion.

Topics: Butadienes; Cell Division; DNA-Binding Proteins; Endometrial Neoplasms; Enzyme Inhibitors; ets-Domain Protein Elk-1; Female; Fibroblast Growth Factor 10; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Gene Expression; Humans; MAP Kinase Signaling System; Nitriles; Phosphorylation; Proto-Oncogene Proteins; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Fibroblast Growth Factor; Stromal Cells; Transcription Factors; Tumor Cells, Cultured