adrenomedullin and Ovarian-Neoplasms

Adrenomedullin (ADM) is a 52-amino acid peptide with structural homology to calcitonin gene-related peptide (CGRP) initially isolated from human pheochromocytoma. ADM is synthesized and is secreted from many mammalian tissues, including the adrenal medulla, endothelial and vascular smooth muscle cells, as well as the myocardium and central nervous system. ADM has been implicated as a mediator of several diseases such as cardiovascular and renal disorders, sepsis, inflammation, diabetes and cancer. ADM is also expressed in a variety of tumors, including breast, endometrial and prostate cancer. ADM has been shown to be a mitogenic factor capable of stimulating growth of several cancer cell types. In addition, ADM is a survival factor for certain cancer cells and an indirect suppressor of the immune response. ADM plays an important role in environments subjected to low oxygen tension, which is a typical feature of solid tumors. Under these conditions, ADM is up regulated and acts as a potent angiogenic factor promoting neovascularization. The major focus of this review will be on the role of ADM in cancer, with emphasis on its utility in diagnostic and prognostic terms, along with its relevance as a therapeutic target.

Topics: Adrenomedullin; Animals; Disease Progression; Endometrial Neoplasms; Female; Humans; Male; Neoplasms; Neovascularization, Physiologic; Ovarian Neoplasms; Pancreatic Neoplasms; Prognosis; Prostatic Neoplasms; Protein Biosynthesis; Receptors, Adrenomedullin; Receptors, Peptide; Signal Transduction

Adrenomedullin (ADM) is a multi-functional peptide related to many kinds of tumors. This study was aimed to investigate the role of ADM on angiogenesis in epithelial ovarian cancer (EOC) and its possible mechanism. The expressions of ADM, vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1α (HIF-1α) and CD34 were examined by immunohistochemistry staining. The relationship among ADM, HIF-1α, VEGF and micro-vessel density (MVD) was assessed in 56 EOC tissues. CAOV3 cells were stably transfected with pcDNA-ADM (plasmid overexpressing ADM gene) or pRNA-shADM (small interfering RNA for ADM gene). Real-time PCR and western blot analysis were performed to detect the expressions of HIF-1α and VEGF. The MTT, transwell migration assay and in vitro tube formation analysis were used to evaluate the proliferation, migration, and tube formation ability of human umbilical vein endothelial cells (HUVECs) which were pretreated with ADM or ADM receptor antagonist ADM22-52. Our findings showed that ADM expression was positively correlated with the expressions of HIF-1α, VEGF or MVD in EOC. ADM upregulated expression of HIF-1α and VEGF in CAOV3 cells. ADM promoted HUVECs proliferation, migration and tube formation. In conclusion, ADM was an upstream molecule of HIF-1α/VEGF and it promoted angiogenesis through upregulating HIF-1α/VEGF in EOC.

Topics: Adrenomedullin; Antigens, CD34; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Microvessels; Middle Aged; Neoplasms, Glandular and Epithelial; Neovascularization, Pathologic; Neovascularization, Physiologic; Ovarian Neoplasms; Peptide Fragments; Up-Regulation; Vascular Endothelial Growth Factor A

Adrenomedullin (AM), a potent vasodilator peptide, presents in various kinds of tumors and promotes angiogenesis. We have previously reported that AM is expressed in epithelial ovarian carcinoma tissue. Here, we investigated the hypothesis that AM might regulate production of vascular endothelial growth factor (VEGF) in epithelial ovarian carcinoma and further promote angiogenic processes.. The messenger RNA expression of VEGF in human epithelial ovarian carcinoma cells (HO-8910) was examined by real-time polymerase chain reaction. Transcriptional control was analyzed by transient transfection assay of VEGF promoter-luciferase hybrid genes and chromatin immunoprecipitation assay. Activation of c-Jun N-terminal kinase (JNK) was detected by Western blotting. The formation of capillarylike structures by EA.hy926 cells cocultured with HO-8910 cells on Matrigel was also studied.. We found that in HO-8910 cells, AM (10⁻¹⁰ to 10⁻⁷ mol/L) enhanced VEGF messenger RNA expression in a time- and concentration-dependent manner, as well as promoter activity. Furthermore, JNK was activated by AM stimulation. The AM-induced increase in VEGF expression was significantly attenuated by SP600125, a specific JNK inhibitor. Chromatin immunoprecipitation assay and promoter activity analysis showed that VEGF expression induced by AM required the activator protein 1 motif on the VEGF promoter. In an in vitro angiogenesis system for endothelial cells (EA.hy926) cocultured with HO-8910 cells, we observed that the addition of AM stimulated endothelial cell tube formation, which could be abolished by VEGF neutralizing antibody.. Our findings suggest that the JNK/Activator protein 1 pathway is involved in AM-induced VEGF expression in HO-8910 cells.

Topics: Adrenomedullin; Antihypertensive Agents; Blotting, Western; Carcinoma, Ovarian Epithelial; Chromatin Immunoprecipitation; Female; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Neoplasms, Glandular and Epithelial; Neovascularization, Pathologic; Ovarian Neoplasms; Real-Time Polymerase Chain Reaction; Regulatory Elements, Transcriptional; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transcription Factor AP-1; Transcriptional Activation; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A

Adrenomedullin (AM), a potent vasodilator peptide, is present in various types of tumors. Here, we constructed short hairpin RNA (shRNA) in order to target the AM gene in vitro using RNA interference (RNAi) technology. HO8910 ovarian cancer cells were transfected, and the effects of AM on proliferation and chemosensitivity of the cells were examined. RT-PCR, real-time PCR and western blot analysis were performed to detect the AM gene and protein expression. The MTT assay was used to observe the effect of AM on proliferation and chemosensitivity of the cells. Also, the protein levels of bcl-2 and the extracellular regulated protein kinase (ERK) were evaluated by western blot analysis. We found that silencing of the AM gene inhibited the proliferation and increased the chemosensitivity of HO8910 cells, reduced the expression of AM mRNA and protein as well as downregulated bcl-2 and p-ERK expression. We, therefore, conclude that silencing of the AM gene in HO8910 ovarian cancer cells inhibited the proliferation and increased the chemosensitivity of the cells through downregulation of ERK and bcl-2 expression. Thus, anti-AM treatment together with suppression of ERK and bcl-2 expression provides a novel research approach for ovarian cancer.

Topics: Adrenomedullin; Antineoplastic Agents; Base Sequence; Carboplatin; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression; Gene Order; Gene Silencing; Genetic Vectors; Humans; MAP Kinase Signaling System; Molecular Sequence Data; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering

Epithelial ovarian cancer (EOC) is one of the leading causes of cancer deaths in women worldwide. Adrenomedullin (AM) is a multifunctional peptide which presents in various kinds of tumors.. In this study, we characterized the expression and function of AM in epithelial ovarian cancer using immunohistochemistry staining. Exogenous AM and small interfering RNA (siRNA) specific for AM receptor CRLR were treated to EOC cell line HO8910. Wound healing assay and flow cytometry were used to measure the migration ability and expression of integrin α5 of HO8910 cells after above treatments. Western blot was used to examine the phosphorylation of FAK and paxillin.. We found that patients with high AM expression showed a higher incidence of metastasis, larger residual size of tumors after cytoreduction and shorter disease-free and overall survival time. Exogenous AM induced ovarian cancer cell migration in time- and dose- dependent manners. AM upregulated the expression of integrin α5 and phosphorylation of FAK, paxillin as well.. Our results suggested that AM contributed to the progression of EOC and had additional roles in EOC cell migration by activating the integrin α5β1 signaling pathway. Therefore, we presumed that AM could be a potential molecular therapeutic target for ovarian carcinoma.

Topics: Adrenomedullin; Calreticulin; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Female; Focal Adhesion Kinase 1; Gene Expression; Humans; Integrin alpha5beta1; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Paxillin; Phosphorylation; Prognosis; Signal Transduction

Stromal elements within a tumor interact with cancer cells to create a microenvironment that supports tumor growth and survival. Adrenomedullin (ADM) is an autocrine/paracrine factor produced by both stromal cells and cancer cells to create such a microenvironment. During differentiation of macrophages, ADM is produced in response to pro-inflammatory stimuli and hypoxia. In this study we investigated the role of ADM as a growth factor for ovarian cancer cells and as a modulator of macrophages. We also analyzed ADM expression levels in a retrospective clinical study using nanofluidic technology and assessment of ADM at the gene level in 220 ovarian cancer patients. To study the effects of ADM, ovarian cancer cell lines A2780, OVCAR-3, and HEY and their drug-resistant counterparts were used for proliferation assays, while monocytes from healthy donors were differentiated in vitro. ADM was a weak growth factor, as revealed by proliferation assays and cell cycle analysis. After culturing cancer cells under stressing conditions, such as serum starvation and/or hypoxia, ADM was found to be a survival factor in HEY but not in other cell lines. In macrophages, ADM showed activity on proliferation/differentiation, primarily in type 2 macrophages (M2). Unexpectedly, the clinical study revealed that high expression of ADM was linked to positive outcome and to cancer with low Ca125. In conclusion, although in vitro ADM was a potential factor in biological aggressiveness, this possibility was not confirmed in patients. Therefore, use of an ADM antagonist would be inappropriate in managing ovarian cancer patients.

Topics: Adenocarcinoma, Papillary; Adrenomedullin; CA-125 Antigen; Cell Cycle; Cell Differentiation; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Culture Media, Serum-Free; Cytokines; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Macrophages; Membrane Proteins; Middle Aged; Multivariate Analysis; Ovarian Neoplasms; Prognosis; Proportional Hazards Models; Retrospective Studies

The aim of the present study was to characterise the expression pattern of the multifunctional vasoactive peptide adrenomedullin (ADM) in human ovarian tumors, and to find hormonal regulators of ADM expression in human ovaries. The expression of ADM messenger RNA (mRNA) was higher in granulosa cell tumors than in fibrothecomas and normal ovaries, as analysed by Northern blots. In normal ovaries, ADM immunoreactivity was localised in both granulosa and thecal cells. Eight of the 90 granulosa cell tumors (9%) showed moderate and 53 (59%) weak ADM immunoreactivity, whereas 27% (11/41) of the fibrothecomas displayed weak ADM staining. FSH, protein kinase A activator (Bu)(2)cAMP, prostaglandin E(2) (PGE(2)), activin A and the broad protein kinase regulator staurosporine decreased ADM mRNA accumulation in cultured granulosa-luteal cells time- and dose-dependently. FSH, (Bu)(2)cAMP and PGE(2) increased progesterone secretion and the accumulation of the steroidogenic acute regulatory protein mRNA in these cells. In conclusion, ADM is expressed in normal human ovaries and sex cord-stromal tumors, particularly in those of granulosa cell origin. FSH, PGE(2,) (Bu)(2)cAMP and activin A suppress ADM gene expression in granulosa-luteal cells. Expression of ADM in human ovaries and its hormonal regulation in granulosa cells suggests a paracrine role for ADM in ovarian function.

Topics: Adrenomedullin; Cells, Cultured; Female; Gene Expression Regulation, Neoplastic; Granulosa Cell Tumor; Granulosa Cells; Humans; Immunohistochemistry; Luteal Cells; Ovarian Neoplasms; Ovary; Paracrine Communication; Progesterone; RNA, Messenger; Sex Cord-Gonadal Stromal Tumors; Thecoma; Tumor Cells, Cultured

Adrenomedullin (AM), a potent vasodilator peptide, present in various kinds of tumors. Basic fibroblast growth factor (bFGF) has broad biological functions involving in the regulation of cell growth, differentiation and proliferation. Here we investigated whether AM expression could be induced by bFGF in human ovarian epithelial carcinoma cells and explored the mediating mechanism. The expression of AM was examined by real time PCR and Western blotting. Transcriptional control was analyzed by transient transfection assay, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. AM expression was detected in ovarian epithelial carcinoma both in mRNA and protein levels in vivo. AM mRNA expression in CAOV(3) was induced by bFGF in a time- and dose-dependent manner, which could be attenuated by bFGF neutralizing antibody. JNK was activated by bFGF stimulation. The bFGF induced increase in AM expression was significantly attenuated by SP600125, a specific JNK inhibitor. EMSA study, ChIP assay and promoter activity analysis showed that AM expression induced by bFGF requires the AP-1 motif on the AM promoter, which is located at -1174 and -922 bp upstream of the transcription start site. The present study demonstrated that AM is expressed in ovarian epithelial carcinoma tissue and bFGF can induce the expression of AM through the JNK-AP-1 pathway in ovarian epithelial carcinoma cell line CAOV(3).

Topics: Adrenomedullin; Adult; Aged; Anthracenes; Female; Fibroblast Growth Factor 2; Humans; JNK Mitogen-Activated Protein Kinases; Middle Aged; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; RNA, Messenger; Transcription Factor AP-1; Tumor Cells, Cultured; Up-Regulation

Adrenomedullin (ADM) is a potent hypotensive peptide produced by macrophages and endothelial cells during ischemia and sepsis. The molecular mechanisms that control ADM gene expression in tumor cells are still poorly defined. It is known, however, that hypoxia potently increases ADM expression by activation of the transcription factor complex hypoxia inducible factor 1 (HIF-1). Proinflammatory cytokines produced by tumor invading macrophages likewise activate expression of ADM. Herein, we show that apart from hypoxia, the proinflammatory cytokine interleukin 1beta (IL-1beta) induced the expression of ADM mRNA through activation of HIF-1 under normoxic conditions and enhanced the hypoxia-induced expression in the human ovarian carcinoma cell line OVCAR-3. IL-1beta significantly increased accumulation and nuclear translocation of HIF-1alpha under normoxic conditions and amplified hypoxic HIF-1 activation. IL-1beta treatment affected neither HIF-1alpha mRNA levels nor the hydroxylation status of HIF-1alpha and, thus, stability of the protein. Instead cycloheximide effectively prevented the increase in HIF-1alpha protein, indicating a stimulatory effect of IL-1beta on HIF-1alpha translation. Finally, treatment of HIF-1alpha with short interfering RNA revealed a significant role for HIF-1 in the IL-1beta-dependent stimulation of ADM expression.

Topics: Adrenomedullin; Cell Hypoxia; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-1; NF-kappa B; Ovarian Neoplasms; Peptides; RNA, Messenger; RNA, Small Interfering; Transcription Factors

To investigate the expression of adrenomedullin (AM) in benign and malignant ovarian tissues and its correlation with estrogen receptors (ERs) mRNA status.. Ovarian carcinoma cell lines, normal ovaries, serous cysts and cancers were analyzed using real-time polymerase chain reaction (PCR) in order to quantify adrenomedullin and ERs mRNAs expression. Some ovarian samples were submitted to laser-capture microdissection to determine the differential expression of target genes in epithelium and stroma.. Ovarian cancer cells express adrenomedullin mRNA for both the ligand and receptor and produce the peptide. In tumors, the ER alpha/beta ratio was higher than in other tissues. Correlations were found between ER alpha and ER beta mRNA and adrenomedullin mRNA expression in tumors.. Adrenomedullin may be involved in both normal and malignant tissue growth through both vascular and growth factor effects. Because of the correlations with ERs status, there is emerging evidence that ovarian cancer is endocrine-related.

Topics: Adrenomedullin; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression; Humans; Ovarian Cysts; Ovarian Neoplasms; Ovary; Peptides; Polymerase Chain Reaction; Receptors, Estrogen; RNA, Messenger; Tumor Cells, Cultured

Adrenomedullin is a multifunctional regulatory peptide with mitogenic and angiogenic capabilities. Objectives in this study were: (1) to describe the effects of adrenomedullin and anti-adrenomedullin antibodies on ovarian carcinoma cell proliferation, and (2) to assess the estradiol regulation of adrenomedullin metabolism.. We assessed the effects of estradiol, adrenomedullin, and anti-adrenomedullin antibodies on cell growth in three human ovarian cell lines. RT-PCR was used to assess mRNA expression and Western blots to determine protein levels.. Estradiol stimulates BG-1 and PEO4 cells growth but not PEO14 cells. Adrenomedullin mRNA expression and secretion were not under estrogen control. Adrenomedullin and anti-adrenomedullin antibodies had no growth effects in vitro. Adjunction of anti-adrenomedullin antibodies to estradiol-stimulated cells significantly inhibited their growth.. Adrenomedullin metabolism is not under estradiol control. Anti-adrenomedullin antibodies display inhibitory effects on cells having high mitogenic activity. This opens the need for additional search toward in vivo specific immunotherapy.

Topics: Adrenomedullin; Antibodies; Cell Division; Cell Line, Tumor; Estradiol; Female; Humans; Ovarian Neoplasms; Peptides; RNA, Messenger

Adrenomedullin (AM) gene expression was analysed in 60 cases of epithelial ovarian cancer, (29 serous, 14 mucinous, 13 endometrioid, three clear cell, and one undifferentiated) using reverse transcription-polymerase chain reaction (RT-PCR); 10 of the cases were of low malignant potential; 25 were stage I; three were stage II; 27 were stage III; and five were stage IV. The level of AM gene expression was described in terms of the relative yield of the AM gene to the beta2-microglobulin gene. AM gene expression ranged from 0.04 to 1.57 (median 0.36). The association between histological grade and AM gene expression was significant (P: = 0.027), however, the association with other clinico-pathological features, i.e. patients' age at diagnosis, stage of disease, residual tumour mass after initial surgery, and histological subtype were not significant. Survival data were available for all patients and univariate Cox regression analysis showed that the AM gene expression was significantly associated with a poor prognosis (P: = 0.019). Immunohistochemical studies showed that AM was localized in the outer cell membrane or the cytoplasm of the carcinoma cells and in the endothelial cells of the tumour stroma. The AM gene expression level may play a key role in the biology of epithelial ovarian cancer and may define a more aggressive tumour phenotype.

Topics: Adrenomedullin; Adult; Aged; Aged, 80 and over; Female; Humans; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Ovarian Neoplasms; Peptides; Prognosis; Proportional Hazards Models; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric