lhrh--his(5)-trp(7)-tyr(8)- and Adenocarcinoma

Expression of the two gonadotropin-releasing hormone homologue peptides GnRHI and GnRHII and their receptor GnRHR has been demonstrated in a number of malignancies. In hormone-dependent breast cancer, GnRH analogs are used for therapy in premenopausal women. Gene expression of GnRHI, II and R was studied in breast biopsies from primary breast adenocarcinoma obtained from the tumor and the adjacent benign tissue. Levels were evaluated by a multiplex real-time RT-PCR. GnRHI transcripts were detected in 14.7% of the benign and 29.4% malignant biopsies and GnRHII in 21.2% benign and 44.1% malignant biopsies. GnRHR was also more frequent in the malignant (54.2%) than in the benign (24.0%) biopsies, at similar expression levels. No transcripts were detected in biopsies from healthy individuals. There was a strong correlation between the presence of GnRHI and GnRHII transcripts and their receptor in the benign and the malignant biopsies. GnRHI, II and R expression correlated significantly with poor prognosis pathological parameters. Immunohistochemistry for GnRHR revealed expression in malignant cells and in epithelial cells of mammary ducts of the adjacent area with pre-cancerous features. In contrast, GnRH I and II peptides were rarely expressed at low levels in breast cancer cells. In conclusion GnRH peptides and receptor are expressed more frequently in breast tumors than in the adjacent mammary tissue, representing a malignant feature. Their expression correlated to tumor characteristics of poor prognosis and was therefore related to more aggressive malignancies. Concomitant expression of peptides and receptor supports an autocrine/paracrine regulating role.

Topics: Adenocarcinoma; Aged; Biopsy; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Gonadotropin-Releasing Hormone; Humans; Middle Aged; Prognosis; Protein Precursors; Receptors, LHRH; Reference Values

GnRH-II is a potent GnRH subtype involved in modulating OVCAR-3 cell proliferation and the invasive properties of JEG-3 cells, and an atypical cAMP-response element (CRE) in the human GnRH-II promoter influences its activation. We demonstrated that the GnRH-II promoter is activated by 8-bromoadenosine-cAMP in several cell lines including alphaT3, TE671, JEG-3, and OVCAR-3 cells and that cAMP enhances GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Moreover, 8-bromoadenosine-cAMP increases cAMP response element-binding protein (CREB) phosphorylation in JEG-3 and OVCAR-3 cells and augments CBP and CCAAT/enhancer-binding protein (C/EBP)-beta coimmunoprecipitation with phosphorylated CREB (p-CREB) in a temporally defined manner from nuclear extracts. When CREB, CBP, and C/EBPbeta levels were knocked down by small interfering RNA, reductions in any of these transcription factors reduced cAMP-enhanced GnRH-II promoter activity and GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Importantly, chromatin immunoprecipitation assay showed that p-CREB bound the CRE within the endogenous GnRH-II promoter within 1 h and that p-CREB association with C/EBPbeta occurs within 2 h of cAMP stimulation, coincident with the first appearance of C/EBPbeta at the CRE. By contrast, maximum interactions between p-CREB and CBP do not occur until at least 4 h after cAMP stimulation, and this is reflected in the progressive loading of CBP at the CRE at 2-4 h, as demonstrated by chromatin immunoprecipitation. Taken together, these data suggest that p-CREB, C/EBPbeta, and CBP are recruited to the CRE of the GnRH-II promoter in a temporarily defined manner to enhance its transcription in JEG-3 and OVCAR-3 cells in response to cAMP.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenocarcinoma; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Choriocarcinoma; CREB-Binding Protein; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Female; Gene Expression Regulation, Neoplastic; Gonadotropin-Releasing Hormone; Humans; Medulloblastoma; Ovarian Neoplasms; Phosphorylation; Promoter Regions, Genetic; RNA, Messenger; Signal Transduction; Transcription Factors

In our previous studies, we demonstrated that ERK1/2 (extracellular signal-regulated protein kinase) and p38 MAPK (mitogen-activated protein kinase) are required for gonadotropin-releasing hormone (GnRH)-II-induced anti-proliferation of ovarian cancer cells. In the present study, we examined the role of the GnRH-I receptor, as well as the activation of protein kinase C (PKC), in the anti-proliferative effect induced by GnRH-I or II in ovarian cancer cells. Our results demonstrated that Antide, a GnRH-I antagonist, reversed the activation of ERK1/2 induced by GnRH-I or II and abolished the anti-proliferative effect of GnRH-I and II in ovarian cancer cells. Transfection of short-interfering RNA to abrogate the gene expression of the GnRH-I receptor reversed GnRH-I and II-induced anti-proliferation. These results indicate that GnRH-I or II induce anti-proliferation through the GnRH-I receptor in ovarian cancer cells. In addition, the activation of ERK1/2 by GnRH-I or II was mimicked by phorbol-12-myristate 13-acetate, a PKC activator. Pretreatment with GF109203X, an inhibitor of PKC, blocked GnRH-induced ERK1/2 activation and anti-proliferation. These results suggest that the activation of PKC is responsible for GnRH-induced ERK1/2 activation and anti-proliferation in ovarian cancer cells. Taken together, these results indicate that binding of GnRH-I and II to the GnRH-I receptor activates ERK1/2 through a PKC-dependent pathway and is essential for GnRH-induced anti-proliferation of ovarian cancer cells.

Topics: Adenocarcinoma; Cell Proliferation; Enzyme Activation; Enzyme Inhibitors; Female; Gonadotropin-Releasing Hormone; Humans; Indoles; Maleimides; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Oligopeptides; Ovarian Neoplasms; Protein Kinase C; Receptors, LHRH; RNA, Small Interfering; Signal Transduction; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

Although a novel second form of GnRH (GnRH-II) has been reported to have an antiproliferative effect on gynecologic cancer cells, its biological mechanism remains to be elucidated. We have previously demonstrated that GnRH-II activates p38 MAPK. There is accumulating evidence that activation of MAPKs by GnRH-I and -II is important for cell proliferation, differentiation, and apoptosis. In the present study, we further investigated the involvement of GnRH-II in the inhibition of cell proliferation and activation of ERK1/2 and c-Jun N-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) in ovarian cancer cells, OVCAR-3. The [(3)H]thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that treatment with GnRH-II suppresses cell proliferation of ovarian cancer cells. Western blot analysis demonstrated that ERK1/2 was activated by GnRH-II (100 nm). Moreover, PD98059 (10 mum), an inhibitor of a MAPK/ERK kinase, reversed the activation of ERK1/2 induced by GnRH-II. The activation of ERK1/2 by GnRH-II subsequently phosphorylated Elk-1 as a downstream pathway, which was blocked by PD98059. On the other hand, it is not likely that GnRH-II activates the JNK/SAPK pathway. Taken together, these results indicate that the ERK1/2 pathway is involved in the effect of GnRH-II on antiproliferation and may be an important target for ovarian cancer therapy.

Topics: Adenocarcinoma; Cell Division; Cell Line, Tumor; DNA-Binding Proteins; ets-Domain Protein Elk-1; Female; Gonadotropin-Releasing Hormone; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Ovarian Neoplasms; Proto-Oncogene Proteins; Transcription Factors

Recent results indicate that a novel second form of GnRH, GnRH-II, has an antiproliferative effect on ovarian and endometrial cancer cells and might be considered as a possible therapy for gynecological tumors. However, the mechanism of the GnRH-II-induced antiproliferative effect is not known. The p38 MAPK, one of the stress-activated protein kinases, is activated by diverse cellular stress and proinflammatory cytokines. In this study, the effect of GnRH-II on the activation of p38 MAPK was investigated, and its possible role in the regulation of cell proliferation and apoptosis was further examined in the human ovarian cancer cell line, OVCAR-3. Treatment with GnRH-II (100 nM) resulted in an activation of p38 MAPK in a time-dependent manner. A significant activation of p38 MAPK was observed at 2, 5, 10, and 15 min after GnRH-II treatment. The activation of p38 MAPK by GnRH-II was reversed in the presence of a specific inhibitor of p38 MAPK, SB203580 (1 microM). The transcription factor, activator protein-1, was activated (1.5-fold) by GnRH-II and attenuated in the presence of SB203580 (1 microM). Treatment with GnRH-II (1 nM, 100 nM, 10 microM) for 2, 4, and 6 d resulted in an inhibition of cell growth in OVCAR-3 cells as determined by thymidine incorporation assay. The effect of GnRH-II (100 nM) on cell proliferation was blocked by pretreatment with SB203580 (1 microM). Furthermore, a significant increase of apoptosis (1.6-fold) was observed after GnRH-II treatment, which was also reversed by pretreatment with SB203580 (1 microM). Taken together, these results indicate that p38 MAPK is involved in the GnRH-II-induced inhibition of cell growth through activator protein-1 activation, which may be related to induction of apoptosis in ovarian cancer cells.

Topics: Adenocarcinoma; Apoptosis; Cell Division; Cell Line, Tumor; Enzyme Inhibitors; Female; Gonadotropin-Releasing Hormone; Humans; Imidazoles; Mitogen-Activated Protein Kinases; Ovarian Neoplasms; p38 Mitogen-Activated Protein Kinases; Pyridines; Signal Transduction; Transcription Factor AP-1