epidermal-growth-factor and Ovarian-Neoplasms

Therapeutics targeting the ErbB protein family receptors have not always yielded favorable or successful results in present cancer therapy. This review discusses the possibility of the clinical adaptation of targeting against heparin-binding epidermal growth factor-like growth factor (HB-EGF), one of the ligands of the ErbB system, in ovarian cancer therapy.. We have previously described the results of studies concerning roles of HB-EGF in tumor formation in ovarian cancer. In brief, lisophosphatidic acid (LPA) and HB-EGF are predominantly expressed in advanced ovarian cancer, and LPA-induced, a disintegrin and metalloprotease-mediated ectodomain shedding of HB-EGF was found to be critical to tumor formation. We also noted that exogenous expression of HB-EGF enhanced tumor formation but inhibition blocked both extracellular signal-related kinase and serine/threonine protein kinase activation. Finally we investigated the antitumor effects of CRM197 - a specific HB-EGF inhibitor - on ovarian cancer cells by evaluating human ovarian cancer cell proliferation.. We discuss alternative strategies to develop the chemotherapeutic agent based on targeting ErbB family ligands rather than their receptors. A phase I study of CRM197 for advanced ovarian cancer has already begun, which is the first approved trial of ErbB-ligand-targeted therapy. We also discuss clinical adaptations based on combination of CRM197 with other conventional chemotherapeutic agents.

Topics: Antineoplastic Agents; Bacterial Proteins; Cell Proliferation; Clinical Trials, Phase I as Topic; Epidermal Growth Factor; Female; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lysophospholipids; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Signal Transduction

Ovarian cancer is a highly metastatic disease and the leading cause of death from gynecologic malignancy. Hence, and understanding of the molecular changes associated with ovarian cancer metastasis could lead to the identification of targets for novel therapeutic interventions. The conversion of an epithelial cell to a mesenchymal cell plays a key role both in the embryonic development and cancer invasion and metastasis. Cells undergoing epithelial-mesenchymal transition (EMT) lose their epithelial morphology, reorganize their cytoskeleton and acquire a motile phenotype through the up- and down-regulation of several molecules including tight and adherent junctions proteins and mesenchymal markers. EMT is believed to be governed by signals from the neoplastic microenvironment including a variety of cytokines and growth factors. In ovarian cancer EMT is induced by transforming growth factor-beta (TGF-beta), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and endothelin-1 (ET-1). Alterations in these cellular pathways candidate them as useful target for ovarian cancer treatment.

Topics: Bone Morphogenetic Protein 4; Cadherins; Cell Differentiation; Endothelin-1; Epidermal Growth Factor; Epithelial Cells; Female; Hepatocyte Growth Factor; Humans; Mesoderm; Neoplasm Metastasis; Ovarian Neoplasms; Transforming Growth Factor beta

The development of the DNA microarray technique facilitated systematic studies of the modulation of gene function. Considerable attention has been focused on members of the growth factor family to elucidate the main regulators of oocyte maturation and ovarian follicle rupture. Among these growth factors, it was found, both in rodents and in humans, that amphiregulin (Ar) and epiregulin (Ep) of the epidermal growth factor (EGF) family were dramatically up-regulated by gonadotrophins in the intact ovary and in primary granulosa cells, respectively. Their role in cumulus expansion and oocyte maturation was established in rodents, and their synthesis under LH stimulation in granulosa cells was demonstrated in humans. To be activated, Ar and Ep must be cleaved by a disintegrin and metalloproteinases (ADAMs) family. However, the precise processing of Ar and Ep by the cumulus cells is still obscure. Future investigations using DNA microarray technique may reveal the repertoire of genes activated in Ar- and Ep-stimulated cumulus cells and may help elucidate the molecular basis of ovulation. EFG-like factors are also involved in triggering ovarian cancer The author hypothesized that the normal ovary maintains cyclicity in the formation of these growth factors preventing the ovary from developing ovarian cancer In ovarian cancer these growth factors are continuously formed in an autocrine manner, leading to transformation and subsequently to ovarian cancer. These growth factors are essential for both normal and neoplastic transformation of the ovary. Taking into consideration these growth factors in the treatment of ovarian malfunction may be one way of curing ovarian cancer.

Topics: Amphiregulin; Animals; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; Female; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Oligonucleotide Array Sequence Analysis; Oocytes; Ovarian Neoplasms; Ovary; Ovulation; Rodentia

Topics: Carcinoma; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-1; Humans; Ligands; Neoplasm Metastasis; Neoplasm Proteins; Ovarian Neoplasms; Prognosis

HB-EGF, a member of the EGF family of growth factors, exerts its biological activity through activation of the EGFR and other ErbB receptors. HB-EGF participates in diverse biological processes, including heart development and maintenance, skin wound healing, eyelid formation, blastocyst implantation, progression of atherosclerosis and tumor formation, through the activation of signaling molecules downstream of ErbB receptors and interactions with molecules associated with HB-EGF. Recent studies have indicated that HB-EGF gene expression is significantly elevated in many human cancers and its expression level in a number of cancer-derived cell lines is much higher than those of other EGFR ligands. Several lines of evidence have indicated that HB-EGF plays a key role in the acquisition of malignant phenotypes, such as tumorigenicity, invasion, metastasis and resistance to chemotherapy. Studies in vitro and in vivo have indicated that HB-EGF expression is essential for tumor formation of cancer-derived cell lines. CRM197, a specific inhibitor of HB-EGF, and an antibody against HB-EGF are both able to inhibit tumor growth in nude mice. These results indicate that HB-EGF is a promising target for cancer therapy, and that the development of targeting tools against HB-EGF could represent a novel type of therapeutic strategy, as an alternative to targeting ErbB receptors.

Topics: Animals; Cell Movement; Epidermal Growth Factor; Female; Genes, erbB; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Mice; Models, Biological; Neoplasms; Ovarian Neoplasms; Phenotype; Transcriptional Activation

Signal transduction mediated by ErbB/HER receptor tyrosine kinases is crucial for the development and maintenance of epithelial tissues, and aberrant signaling is frequently associated with malignancies of epithelial origin. This review focuses on the roles played by the Hsp90 chaperone machinery in the regulation of signaling through the ErbB/HER network, and discusses potential therapeutic strategies that disrupt chaperone functions. Hsp90 and its associated cochaperones regulate ErbB signal transduction through multiple mechanisms. The chaperone system controls the stability of the nascent forms of both ErbB-1 (EGF-receptor) and ErbB-2/HER2, while regulation of the mature form is restricted to ErbB-2. Regulation by the Hsp90 complex extends to downstream effectors of ErbB signaling, namely Raf-1, Pdk-1 and Akt/PKB. Disrupting the function of Hsp90 results in the degradation of both the receptors and their effectors, thereby inhibiting tumor cell growth. The importance of an Hsp90-recognition motif located within the kinase domain of ErbB-2 is discussed, as well as a direct role for Hsp90 in regulating tyrosine kinase activity. In light of recent observations, we emphasize the ability of specific tyrosine kinase inhibitors to selectively target ErbB-2 to the chaperone-mediated degradation pathway. ErbB-specific drugs are already used to treat cancers, and clinical trials are underway for additional compounds that intercept ErbB signaling, including drugs that target Hsp90. Hence, the dependence of ErbB-2 upon Hsp90 reveals an Achilles heel, which opens a window of opportunity for combating cancers driven by the ErbB/HER signaling network.

Topics: 3-Phosphoinositide-Dependent Protein Kinases; Amino Acid Motifs; Animals; Antineoplastic Agents; Brain Neoplasms; Breast Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Female; Glioma; HSP90 Heat-Shock Proteins; Humans; Neuregulins; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-raf; Receptor, ErbB-2; Signal Transduction; Tumor Cells, Cultured

Topics: Ascitic Fluid; Calcium; Cell Division; Drug Design; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lysophospholipids; Ovarian Neoplasms; Signal Transduction

Topics: Animals; Antineoplastic Agents; Bacterial Proteins; Cell Division; Drug Design; Epidermal Growth Factor; ErbB Receptors; Female; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Lysophospholipids; Ovarian Neoplasms; Signal Transduction

In summary, the EGF/ErbB family of receptor tyrosine kinases has been shown to play a key role in normal ovarian follicle development, and cell growth regulation of the ovarian surface epithelium. Disregulation of these normal growth regulatory pathways, including overexpression and/or mutation of EGFR/ErbB receptor family members, as well as elements of their downstream signalling pathways, have been shown to contribute to the etiology and progression of epithelial ovarian cancer. It is, therefore, not surprising that these gene products, and their related soluble receptor isoforms may have clinical utility as tumor and/or serum biomarkers of disease activity. Moreover, since several of these soluble receptor isoforms have potent growth inhibitory activity, and are naturally occurring in the circulation, they are ideal candidates for the development of novel therapeutics for the treatment of ovarian cancer patients.

Topics: Binding Sites; Biomarkers, Tumor; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation; Genes, erbB; Humans; Ligands; Ovarian Neoplasms; Receptor Protein-Tyrosine Kinases; Signal Transduction; Solubility

As with many other tumors, the origin and development of ovarian cancer is constituted by several molecular mechanisms, many of which are still unknown. Furthermore, data in the literature are incomplete and often contradictory, and they are mainly founded on results obtained on cell lines and not on observations based on the in vivo study of ovarian cancer. Despite this situation, the study of control mechanisms of proliferation and differentiation in normal ovarian functioning has enabled clinicians to identify certain growth factors and oncogenes which seem to have an important role in the neoplastic transformation of ovarian tissue. In this review, our aim is to summarise the most important data regarding function of growth factors and oncogene in normal and neoplastic epithelial ovarian cells.

Topics: Cell Division; Cell Line, Tumor; Epidermal Growth Factor; Female; Growth Substances; Humans; Ovarian Neoplasms; Transforming Growth Factor beta

Topics: Carcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Transforming Growth Factors

Monoclonal antibodies can be coupled with PE to make very potent ITs. Two of these ITs (PE-HB21 and OVB-3-PE) have been shown to have antitumor activity in a nude mouse model of ovarian cancer. PE ITs are at least 10-fold more active than the corresponding RTA IT. Deletion analysis of the structural gene of PE has helped assign specific functions to different portions of the molecule. Current efforts are focused on making ITs with recombinant PE.

Topics: ADP Ribose Transferases; Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Bacterial Toxins; Cross-Linking Reagents; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Exotoxins; Female; Genes, Bacterial; Humans; Immunotoxins; Mice; Mice, Nude; Ovarian Neoplasms; Peptide Elongation Factor 2; Peptide Elongation Factors; Protein Engineering; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Ricin; Transferrin; Virulence Factors

Topics: Aminoacridines; Amsacrine; Animals; Anthracenes; Antineoplastic Agents; Blood Physiological Phenomena; Bone Marrow; Breast Neoplasms; Colony-Forming Units Assay; Drug Evaluation; Drug Evaluation, Preclinical; Drug Stability; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Humans; Interferons; Karyotyping; Leukemia; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Multiple Myeloma; Neoplasm Transplantation; Neoplasms; Ovarian Neoplasms; Platelet-Derived Growth Factor; Suspensions; Transplantation, Heterologous; Tumor Stem Cell Assay

Ovarian cancer is a serious threat to female health, although the incidence of it is relatively low, its mortality rate remains high due to its intense invasion and metastasis. Therefore, it is urgent to explore new treatment strategies for ovarian cancer. In this study, paclitaxel and emodin were encapsulated in different micelles, and loaded on the surface of the micelles with epidermal growth factor (EGF) as the targeting molecule, made compound formulations in proportion. In this study, EGF-modified paclitaxel micelles and EGF-modified emodin micelles were characterized, their inhibitory effects on SKOV3 cell proliferation and invasion were studied

Topics: Cell Line, Tumor; Emodin; Epidermal Growth Factor; Female; Humans; Liposomes; Micelles; Ovarian Neoplasms; Paclitaxel

Regucalcin, which plays a multifunctional role in cell regulation, contributes as a suppressor in carcinogenesis. Survival of cancer patients is prolonged with high expression of regucalcin in tumor tissues. Ovarian cancer is the most lethal in gynecologic malignancies. This study elucidates the repressive role of regucalcin on the growth of human ovarian cancer SK-OV-3 cells that are resistant to cytotoxic cancer drugs.. SK-OV-3 wild type-cells and regucalcin-overexpressing cells (transfectants) were cultured in Dulbecco's Modification of Eagle's Medium containing 10 % fetal bovine serum.. Colony formation and proliferation of SK-OV-3 cells were repressed by regucalcin overexpression. The suppressive effects of regucalcin on proliferation were independent of cell death. The proliferation of SK-OV-3 wild-type cells was repressed by various inhibitors, including cell cycle, signaling processes, and transcriptional activity. The effects of all inhibitors were not revealed in transfectants, suggesting the involvement of multiple signaling pathways in regucalcin effects. Of note, the overexpressed regucalcin declined the levels of Ras, Akt, mitogen-activating protein kinase, NF-κB p65, β-catenin, and STAT3, while it raised the levels of tumor suppressors p53 and Rb, and cell cycle inhibitor p21. Interestingly, the stimulatory effects of epidermal growth factor (EGF) on cell proliferation were blocked in regucalcin-overexpressing cells. Extracellular regucalcin repressed the proliferation independent of the death of SK-OV-3 cells and blocked EGF-enhanced cell proliferation.. The overexpressed regucalcin may repress cell proliferation by targeting diverse signal pathways, including EGF signaling. This study offers a novel approach to the treatment of ovarian cancer with regucalcin.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Female; Humans; Ovarian Neoplasms; Signal Transduction

The type I cGMP-dependent protein kinase (PKG I) is recognized as a tumor suppressor, but its role in EGFR regulated epithelial ovarian cancer (EOC) progression remains unclear. We evaluated the in vivo and in vitro effects of activated PKG I in EGF-induced EOC cell proliferation, migration, and invasion. The expressions of EGFR and PKG I were elevated, but the activated PKG I was decreased in EOC tissues of patients and cells lines. The addition of 8-Br-cGMP, a specific PKG I activator, attenuated the EGF-induced EOC cell proliferation, migration, and invasion in vitro. Similarly, activated PKG I also attenuated EOC progression in vivo using an EOC xenograft nude mouse model. The activated PKG I interacted with EGFR, causing increased threonine (693) phosphorylation and decreased tyrosine (1068) phosphorylation of EGFR, which resulted in disrupted EGFR-SOS1-Grb2 combination. Subsequently, the cytoplasmic phosphorylation of downstream proteins (c-Raf, MEK1/2, and ERK1/2) were declined, impeding the phosphorylated ERK1/2's nucleus translocation, and this reduction of phosphorylated tyrosine (1068) EGFR and ERK1/2 were also abolished by Rp-8-Br-cGMPS. Our results suggest that the activation of PKG I attenuates EGF-induced EOC progression, and the 8-Br-cGMP-PKG I-EGFR/MEK/ERK axis might be a potential target for EOC therapy.

Topics: Animals; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; MAP Kinase Signaling System; Mice; Ovarian Neoplasms; Phosphorylation; Tyrosine

Topics: Adaptor Proteins, Signal Transducing; Epidermal Growth Factor; Female; Humans; Intracellular Signaling Peptides and Proteins; Ovarian Neoplasms; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Signal Transduction

The gene for receptor tyrosine kinase ErbB2 is amplified in breast and ovarian tumours. The linear pathway by which signals are transduced through ErbB2 are well known. However, second generation questions that address spatial aspects of signaling remain. To address this, we have undertaken a mass spectrometry approach to identify phosphoproteins specific for ErbB2 using the inhibitors Lapatinib and CP724714 in ovarian cancer cells. The ErbB2 specific proteins identified in SKOV-3 cells were Myristoylated alanine-rich C-kinase substrate, Protein capicua homolog, Protein peptidyl isomerase G, Protein PRRC2C, Chromobox homolog1 and PRP4 homolog. We have evaluated three phosphoproteins PKM2, Aldose reductase and MARCKS in SKOV-3 cells. We observed that PKM2 was phosphorylated by EGF but was not inhibited by Lapatinib and CP724714. The activity of aldose reductase in reducing NADPH as a substrate was significantly higher in EGF stimulated cells which was inhibited by Lapatinib and CP724714 but not by Geftinib (EGFR inhibitor). MARCKS was phosphorylated on stimulation of SKOV-3 cells with EGF that was inhibited by Lapatinib and CP724714 which was dependent on the kinase activity of ErbB2. These results have identified phosphoproteins that are specific to ErbB2 which have not been previously reported and sets the basis for future experiments.

Topics: Aldehyde Reductase; Cell Line, Tumor; Epidermal Growth Factor; Female; Humans; Lapatinib; Ovarian Neoplasms; Phosphoproteins; Receptor, ErbB-2

In individuals with ovarian cancer, an increase in the circulating level of the epidermal growth factor (EGF) is readily apparent. Ovarian cancer cells exhibit signaling pathway of the epidermal growth factor (EGFR) and respond to the EGF. Annona muricata (AM) has been shown to decrease ovarian cell proliferation however, role of AM in regulating EGF actions is not yet to be reported.. In this study, we proposed that the fractionated compound acetogenin can inhibit the activation of EGFR-regulated signaling cascades such as MAPK7 / PI3K-Akt / mTOR / STAT upon EGF stimulation.. Ethanolic extract was prepared for the whole AM plant and Thin Layer Chromatography (TLC) was performed to characterize the secondary metabolites and each fraction was assessed using kedde reagent for the presence of acetogenin. The effects of acetogenins were then tested on the survival of PA-1 ovarian cancer cells under basal and EGF stimulated conditions. To delineate the role of acetogenin in EGFR signaling cascades, the in silico docking studies were conducted.. The fraction of acetogenin decreased the viability of EGF induced PA-1 ovarian cancer cells that indicating the EGF inhibitory effects of acetogenin. The docking studies specifically illustrated that when the acetogenin binding with tyrosine kinase (TK) and regulatory unit (RU) which subsequently resulted in a reduction in EGF induced the survival of PA-1 ovarian cancer cells.. The vital regulatory role of acetogenin reported in this study indicate significant anticancer activities of acetogenin from AM. The in silico study of the acetogenin function predicted that it binds specifically to Asp837 (phosphor-acceptor site) of EGFR, essential for phosphorylation of substrates in the TK domain and RU which promote downstream signaling.. Acetogenin isolated from AM effectively inhibited the survival of PA-1 ovarian cancer cells through impaired EGF signaling.

Topics: Acetogenins; Annona; Cell Line, Tumor; Epidermal Growth Factor; Female; Humans; Neoplasm Proteins; Ovarian Neoplasms; Signal Transduction

Ovarian cancer (OvCA) is the most lethal neoplasia among gynecologic malignancies and faces high rates of new cases particularly in South America. In special, the High Grade Serous Ovarian Carcinoma (HGSC) presents very poor prognosis with deaths caused mainly by metastasis. Among several mechanisms involved in metastasis, the Epithelial to Mesenchymal Transition (EMT) molecular reprogramming represents a model for latest stages of cancer progression. EMT promotes important cellular changes in cellular adhesion and cell-cell communication, which particularly depends on the paracrine signaling from neighbor cells. Considering the importance of cellular communication during EMT and metastasis, here we analyzed the changes in the secretome of the ovarian cancer cell line Caov-3 induced to EMT by Epidermal Growth Factor (EGF). Using a combination of GEL-LC-MS/MS and stable isotopic metabolic labelling (SILAC), we identified up-regulated candidates during EMT as a starting point to identify relevant proteins for HGSC. Based on public databases, our candidate proteins were validated and prioritized for further analysis. Importantly, several of the protein candidates were associated with cellular vesicles, which are important to the cell-cell communication and metastasis. Furthermore, the association of candidate proteins with gene expression data uncovered a subset of proteins correlated with the mesenchymal subtype of ovarian cancer. Based on this relevant molecular signature for aggressive ovarian cancer, supported by protein and gene expression data, we developed a targeted proteomic method to evaluate individual OvCA clinical samples. The quantitative information obtained for 33 peptides, representative of 18 proteins, was able to segregate HGSC from other tumor types. Our study highlighted the richness of the secretome and EMT to reveal relevant proteins for HGSC, which could be used in further studies and larger patient cohorts as a potential stratification signature for ovarian cancer tumor that could guide clinical conduct for patient treatment.

Topics: Biomarkers, Tumor; Cell Communication; Cell Line, Tumor; Chromatography, Liquid; Cystadenocarcinoma, Serous; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Isotope Labeling; Neoplasm Invasiveness; Neoplasm Staging; Ovarian Neoplasms; Protein Interaction Maps; Proteomics; Tandem Mass Spectrometry; Up-Regulation

The epidermal growth factor receptor (EGFR) is overexpressed in many types of cancer, including epithelial ovarian cancer (EOC), and its expression has been found to correlate with advanced stage and poor prognosis. The EGFR ligand amphiregulin (AREG) has been investigated as a target for human cancer therapy and is known to have an autocrine role in many cancers. A cytokine array identified AREG as one of several cytokines upregulated by EGF in a phosphatidylinositol 3-kinase (PI3-K) dependent manner in EOC cells. To investigate the functional role of AREG in EOC, its effect on cellular migration and proliferation was assessed in two EOC cells lines, OV167 and SKOV3. AREG increased both migration and proliferation of EOC cell line models through activation of PI3-K signaling, but independent of mitogen activated protein kinase (MAPK) signaling. Through an AREG autocrine loop mediated via PI3-K, upregulation of AREG led to increased levels of both AREG transcript and secreted AREG, while downregulation of endogenous AREG decreased the ability of exogenous AREG to induce cell migration and proliferation. Further, inhibition of endogenous AREG activity or metalloproteinase activity decreased EGF-induced EOC migration and proliferation, indicating a role for soluble endogenous AREG in mediating the functional effects of EGFR in inducing migration and proliferation in EOC.

Topics: Amphiregulin; Autocrine Communication; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Up-Regulation

Anoikis acts as a critical barrier to metastasis by inducing cell death upon cancer cell detachment from the extracellular matrix (ECM), thereby preventing tumor cell dissemination to secondary sites. The induction of anoikis requires the lysosomal-mediated downregulation of epidermal growth factor receptors (EGFRs) leading to termination of pro-survival signaling. In this study, we demonstrate that depletion of pre-mRNA splicing factor 4 kinase (PRP4K; also known as PRPF4B) causes dysregulation of EGFR trafficking and anoikis resistance. We also report a novel cytoplasmic localization of PRP4K at the late endosome, and demonstrate both nuclear and cytoplasmic localization in breast, lung and ovarian cancer tissue. Mechanistically, depletion of PRP4K leads to reduced EGFR degradation following cell detachment from the ECM and correlates with increased TrkB, vimentin and Zeb1 expression. As a result, PRP4K loss promotes sustained growth factor signaling and increased cellular resistance to anoikis in vitro and in a novel zebrafish xenotransplantation model of anoikis sensitivity, as well as increased metastasis in a mouse model of ovarian cancer. Thus, PRP4K may serve as a potential biomarker of anoikis sensitivity in ovarian and other epithelial cancers.

Topics: Animals; Anoikis; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Down-Regulation; Endosomes; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Humans; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Ovarian Neoplasms; Protein Serine-Threonine Kinases; Ribonucleoprotein, U4-U6 Small Nuclear; RNA, Small Interfering; Signal Transduction; Xenograft Model Antitumor Assays; Zebrafish

The recurrence, metastasis and poor prognosis are important characteristics of ovarian carcinoma (OC), which are associated with exfoliation of cells from the primary tumor and colonization of the cells in pelvic cavity. On the other hand, the life quality of the patients undergoing surgical resection of OC was influenced by postoperative adhesions. Therefore, preventing postoperative implant tumor and adhesion may be effective methods to improve OC treatment. HyaRegen Gel, a cross-linked hyaluronan gel (CHAG), has been widely used as an anti-adhesive agent following pelvic operation in clinic. However, whether it can affect the implantation and growth of OC cells or not is still not clear.. Migration and invasion assays were applied to detect the effect of CHAG on migration and invasion of OC cells. Western blotting was performed to detect the phosphorylation/activation of EGFR and ERK, and the expression of PCNA and MMP7. Pull down assay was used to analyze the effect of CHAG on the activation of small G protein Rac1. Nude mice implantation tumor model was applied to observe the effect of CHAG on implantation tumor of OC cells.. The results of in vitro experiments showed that CHAG suppressed both basic and EGF-induced migration and invasion of OC cells, blocked the activation of EGF-initiated EGFR activation, inhibited downstream signal transduction of EGFR, and decreased expression of proliferation and migration/invasion related proteins. Meanwhile, results of in vivo experiments showed that CHAG not only inhibited the formation of implantation tumor of OC cells but also delayed the of the growth of the tumors.. CHAG inhibited migration, invasion and proliferation of OC cells in vitro, and suppressed development of implantation tumor of OC in vivo. This made it as both anti-tumor and anti-adhesion agents.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Female; Gels; Humans; Hyaluronic Acid; Mice; Ovarian Neoplasms; rac1 GTP-Binding Protein; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

A recent paradigm shift in ovarian cancer research is the finding that many ovarian cancers may originate from fallopian tube epithelial (FTE) cells. As tissue stem and progenitor cells often serve as cells of origin of cancer, a better understanding of FTE stem/progenitor cells and how they become transformed is essential for early detection and prevention of ovarian cancer. To facilitate study of FTE stem/progenitor cells in model systems, we established an organoid culture system for mouse FTE cells. We find that EPCAM+ mouse FTE cells can be stably cultured long-term under a minimal condition of activated EGF signaling and suppressed TGFbeta signaling. We show that both Notch and Wnt signaling are required for growth of FTE cells in organoids, and further activation of Wnt signaling supports their maturation toward the ciliated cell lineage. Lastly, by analyzing the frequency of organoid-forming cells in different portions of the fallopian tube (FT), we find that the distal portion of the FT, which includes the fimbria, is enriched with organoid-forming FTE stem cells.

Topics: Animals; Epidermal Growth Factor; Epithelial Cells; Fallopian Tubes; Female; Mice; Organoids; Ovarian Neoplasms; Receptors, Notch; Stem Cells; Transforming Growth Factor beta; Wnt Signaling Pathway

Resveratrol, a naturally occurring phytoalexin, has long been known to play an important regulatory role in key functions in cell physiology. This multifunctional role of resveratrol is explained by its ability to interact with several targets of various cell pathways. In the recent past, synthetic chemical modifications have been made in an attempt to enhance the biological effects of resveratrol, including its anti-cancer properties. In this study, we investigated the molecular mechanisms of action of novel trans-restricted analogues of resveratrol in which the C-C double bond of the natural derivative has been replaced by diaryl-substituted imidazole analogues. In ovarian cancer models, the results of in vitro screening revealed that the resveratrol analogues exhibited enhanced anti-proliferative properties compared with resveratrol. We found that the resveratrol analogues also significantly inhibited Akt and MAPK signalling and reduced the migration of IL-6 and EGF-treated cells. Finally, in ascite-derived cancer cells, we demonstrated that the resveratrol analogues reduced the expression of epithelial mesenchymal transition (EMT) markers. Collectively, these findings indicate the enhanced anti-cancer properties of the resveratrol analogues.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Female; Humans; Interleukin-6; Ovarian Neoplasms; Resveratrol; Signal Transduction; Stilbenes

Cluster of differentiation (CD) 44 and epidermal growth factor (EGF) are closely involved in cellular migration and have been used as stem cell markers. Although the hyaluronan (HA)‑binding CD44 is responsible for enhanced cellular motility, the mechanism underlying its actions in various cell types and clinical conditions have yet to be elucidated. In the present study, the multikinase inhibitor sorafenib was used to investigate the diverse effects of EGF stimulation on epithelial‑mesenchymal transition (EMT) in ovarian cancer cells using immunoblotting and reverse transcription‑polymerase chain reaction. In addition, the association between EGF and CD44/HA signaling pathways in the control of mesenchymal phenotype was determined by gene silencing with small interfering RNA transfection. EGF stimulation of ovarian cancer cells increased cellular migration, mesenchymal transition, CD44 expression and the activation of matrix metalloproteinase (MMP)‑2 and MMP‑9. Sorafenib effectively suppressed the loss of epithelial characteristics in EGF‑treated SK‑OV‑3 ovarian cancer cells, via targeting the mitogen‑activated protein kinase (MAPK)/extracellular signal‑regulated kinase (ERK) pathway. Although treatment of Caov‑3 ovarian cancer cells with sorafenib blocked the expression of mesenchymal phenotypes following EGF stimulation, EGF‑activated Caov‑3 cells exhibited reduced MAPK/ERK signaling. Furthermore, EGF‑activated Caov‑3 cells increased the expression of hyaluronan synthase 2 and HA‑CD44 ligation in EGF‑exposed Caov‑3 cells, which resulted in the activation of the Ras/Raf/MEK signaling pathway, amplification of migratory activity and the expression of mesenchymal markers, including N‑cadherin and vimentin. Furthermore, silencing EGFR in SK‑OV‑3 cells and CD44 in Caov‑3 cells suppressed their migratory activity, through inhibition of the MAPK/ERK pathway. The present results suggested that EGF‑mediated signaling may regulate metastasis and invasion of ovarian cancer cells, in a cancer cell type‑dependent manner.

Topics: Basigin; Cell Line, Tumor; Cell Movement; Down-Regulation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Female; Humans; Hyaluronan Receptors; Hyaluronan Synthases; Hyaluronic Acid; MAP Kinase Signaling System; Neoplasm Invasiveness; Niacinamide; Ovarian Neoplasms; Phenotype; Phenylurea Compounds; RNA, Small Interfering; Signal Transduction; Sorafenib

Epithelial to mesenchymal transition (EMT) is a well-orchestrated process that culminates with loss of epithelial phenotype and gain of a mesenchymal and migratory phenotype. EMT enhances cancer cell invasiveness and drug resistance, favoring metastasis. Dysregulation of transcription factors, signaling pathways, miRNAs and growth factors including EGF, TGF-beta and HGF can trigger EMT. In ovarian cancer, overexpression of the EGFR family is associated with more aggressive clinical behavior. Here, the ovarian adenocarcinoma cell line Caov-3 was induced to EMT with EGF in order to identify specific mechanisms controlled by this process. Caov-3 cells induced to EMT were thoroughly validated and a combination of subcellular proteome enrichment, GEL-LC-MS/MS and SILAC strategy allowed consistent proteome identification and quantitation. Protein network analysis of differentially expressed proteins highlighted regulation of metabolism and cell cycle. Activation of relevant signaling pathways, such as PI3K/Akt/mTOR and Ras/Erk MAPK, in response to EGF-induced EMT was validated. Also, EMT did not affected the proliferation rate of Caov-3 cells, but led to cell cycle arrest in G1 phase regulated by increased levels of p21Waf1/Cip1, independently of p53. Furthermore, a decrease in G1 and G2 checkpoint proteins was observed, supporting the involvement of EGF-induced EMT in cell cycle control.. Cancer is a complex multistep process characterized by accumulation of several hallmarks including epithelial to mesenchymal transition (EMT), which promotes cellular and microenvironmental changes resulting in invasion and migration to distant sites, favoring metastasis. EMT can be triggered by different extracellular stimuli, including growth factors such as EGF. In ovarian cancer, the most lethal gynecological cancer, overexpression of the EGFR family is associated with more aggressive clinical behavior, increasing mortality rate caused by metastasis. Our proteomic data, together with specific validation of specific cellular mechanisms demonstrated that EGF-induced EMT in Caov-3 cells leads to important alterations in metabolic process (protein synthesis) and cell cycle control, supporting the implication of EGF/EMT in cancer metastasis, cancer stem cell generation and, therefore, poor prognosis for the disease.

Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Chromatography, Liquid; Cyclin-Dependent Kinase Inhibitor p21; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Female; G1 Phase Cell Cycle Checkpoints; Humans; Neoplasm Invasiveness; Ovarian Neoplasms; Proteomics; Tandem Mass Spectrometry

Connexin43 (Cx43) has been shown to regulate cell proliferation and its downregulation is correlated with poor prognosis and survival in several types of human cancer. Cx43 expression levels are frequently downregulated in human ovarian cancer, suggesting a potential role for Cx43 in regulating the progression of this disease. Epidermal growth factor (EGF) is a well-characterized hormone that stimulates ovarian cancer cell proliferation. Although EGF is able to regulate Cx43 expression in other cell types, it is unclear whether EGF can regulate Cx43 expression in ovarian cancer cells. Additionally, it remains unknown whether Cx43 is involved in EGF-stimulated ovarian cancer cell proliferation. In the present study, we demonstrate that treatment with EGF upregulates Cx43 expression in two ovarian cancer cell lines, SKOV3 and OVCAR4. Although treatment with EGF activates both ERK1/2 and Akt signaling pathways, pharmacological inhibition and siRNA-mediated knockdown suggest that only the activation of Akt1 is required for EGF-induced Cx43 upregulation. Functionally, Cx43 knockdown enhanced basal and EGF-induced cell proliferation, whereas the proliferative effects of EGF were reduced by Cx43 overexpression. Co-treatment with the gap junction inhibitor carbenoxolone did not alter the suppressive effects of Cx43 overexpression on EGF-induced cell proliferation, suggesting a gap junction-independent mechanism. This study reveals an important role for Cx43 as a negative regulator of EGF-induced human ovarian cancer cell proliferation.

Topics: Cell Communication; Cell Line, Tumor; Cell Proliferation; Connexin 43; Epidermal Growth Factor; Female; Gap Junctions; Humans; Ovarian Neoplasms; Signal Transduction

Many cancers are dependent on inappropriate activation of epidermal growth factor receptor (EGFR), and drugs targeting this receptor can improve patient survival, although benefits are generally short-lived. We reveal a novel mechanism linking EGFR and the membrane-spanning, cancer-promoting protein CDCP1 (CUB domain-containing protein 1). Under basal conditions, cell surface CDCP1 constitutively internalizes and undergoes palmitoylation-dependent degradation by a mechanism in which it is palmitoylated in at least one of its four cytoplasmic cysteines. This mechanism is functional in vivo as CDCP1 is elevated and palmitoylated in high-grade serous ovarian tumors. Interestingly, activation of the EGFR system with EGF inhibits proteasome-mediated, palmitoylation-dependent degradation of CDCP1, promoting recycling of CDCP1 to the cell surface where it is available to mediate its procancer effects. We also show that mechanisms inducing relocalization of CDCP1 to the cell surface, including disruption of its palmitoylation and EGF treatment, promote cell migration. Our data provide the first evidence that the EGFR system can function to increase the lifespan of a protein and also promote its recycling to the cell surface. This information may be useful for understanding mechanisms of resistance to EGFR therapies and assist in the design of treatments for EGFR-dependent cancers.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Antigens, Neoplasm; Cell Adhesion Molecules; Cell Line, Tumor; Cell Membrane; Cell Movement; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Interleukin-6; Lipoylation; Membrane Proteins; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Proteins; Neoplasm Transplantation; Ovarian Neoplasms; Protein Transport; Transplantation, Heterologous; Tumor Necrosis Factor-alpha

Sprouty (SPRY) proteins are well-characterized factors that inhibit receptor tyrosine kinase signaling. Our Human Exonic Evidence-Based Oligonucleotide (HEEBO) microarray results showed that the mRNA levels of SPRY2, but not of SPRY1 or SPRY4, are down-regulated in high-grade serous ovarian carcinoma (HGSC) tissues and epithelial ovarian cancer (EOC) cell lines. Molecular inversion probe (MIP) copy number analysis showed the deletion of the SPRY2 locus in HGSC. Overexpression of SPRY2 reduced EGF-induced cell invasion by attenuating EGF-induced E-cadherin down-regulation. Moreover, a positive correlation between SPRY2 and E-cadherin protein levels was observed in HGSC tissues. This study reveals the loss of SPRY2 in HGSC and indicates an important tumor-suppressive role for SPRY2 in mediating the stimulatory effect of EGF on human EOC progression.

Topics: Cadherins; Carcinoma; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Neoplasm Staging; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Signal Transduction

Although epithelial cell adhesion molecule (EpCAM) is overexpressed in human epithelial ovarian cancer (EOC), some contradictory results have been reported regarding the correlation between EpCAM overexpression and patient survival. In addition to this controversy, the function and regulation of EpCAM in EOC remain largely unknown. Here, we show that epidermal growth factor (EGF) up-regulates EpCAM expression by activating ERK1/2 signaling in a human EOC cell line, SKOV3. Additionally, EpCAM overexpression suppresses not only basal but also EGF-stimulated SKOV3 cell migration, whereas EpCAM knockdown increases both basal and EGF-stimulated cell migration in another human EOC cell line, OVCAR4. This study demonstrates the regulation of EpCAM and its role in mediating the effects of EGF on human EOC cell migration.

Topics: Antigens, Neoplasm; Cell Adhesion Molecules; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelial Cell Adhesion Molecule; ErbB Receptors; Female; Gene Knockdown Techniques; Humans; MAP Kinase Signaling System; Ovarian Neoplasms; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Up-Regulation

Cranberry flavonoids (flavonols and flavan-3-ols), in addition to their antioxidant properties, have been shown to possess potential in vitro activity against several cancers. However, the difficulty of isolating cranberry compounds has largely limited anticancer research to crude fractions without well-defined compound composition. In this study, individual cranberry flavonoids were isolated to the highest purity achieved so far using gravity and high performance column chromatography and LC-MS characterization. MTS assay indicated differential cell viability reduction of SKOV-3 and OVCAR-8 ovarian cancer cells treated with individual cranberry flavonoids. Treatment with quercetin aglycone and PAC DP-9, which exhibited the strongest activity, induced apoptosis, led to caspase-3 activation and PARP deactivation, and increased sensitivity to cisplatin. Furthermore, immunofluorescence microscopy and western blot study revealed reduced expression and activation of epidermal growth factor receptor (EGFR) in PAC DP-9 treated SKOV-3 cells. In addition, quercetin aglycone and PAC DP-9 deactivated MAPK-ERK pathway, induced downregulation of cyclin D1, DNA-PK, phospho-histone H3 and upregulation of p21, and arrested cell cycle progression. Overall, this study demonstrates promising in vitro cytotoxic and anti-proliferative properties of two newly characterized cranberry flavonoids, quercetin aglycone and PAC DP-9, against ovarian cancer cells.

Topics: Antineoplastic Agents; Antioxidants; Blotting, Western; Carcinoma, Ovarian Epithelial; Caspase 3; Cell Cycle Checkpoints; Cell Survival; Chromatography; Cisplatin; DNA Fragmentation; Drug Synergism; Epidermal Growth Factor; Fluorescent Antibody Technique, Indirect; Humans; In Situ Nick-End Labeling; Mitogen-Activated Protein Kinase Kinases; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Proanthocyanidins; Quercetin; Tumor Cells, Cultured; Vaccinium macrocarpon

Several clinical trials in oncology have reported increased mortality or disease progression associated with erythropoiesis-stimulating agents. One hypothesis proposes that erythropoiesis-stimulating agents directly stimulate tumor proliferation and/or survival through cell-surface receptors. To test this hypothesis and examine if human tumors utilize the erythropoietin receptor pathway, the response of tumor cells to human recombinant erythropoietin was investigated in disaggregated tumor cells obtained from 186 patients with colorectal, breast, lung, ovarian, head and neck, and other tumors. A cocktail of well characterized tumor growth factors (EGF, HGF, and IGF-1) were analyzed in parallel as a positive control to determine whether freshly-isolated tumor cells were able to respond to growth factor activation ex vivo. Exposing tumor cells to the growth factor cocktail resulted in stimulation of survival and proliferation pathways as measured by an increase in phosphorylation of the downstream signaling proteins AKT and ERK. In contrast, no activation by human recombinant erythropoietin was observed in isolated tumor cells. Though tumor samples exhibited a broad range of cell-surface expression of EGFR, c-Met, and IGF-1R, no cell-surface erythropoietin receptor was detected in tumor cells from the 186 tumors examined (by flow cytometry or Western blot). Erythropoiesis-stimulating agents did not act directly upon isolated tumor cells to stimulate pathways known to promote proliferation or survival of human tumor cells isolated from primary and metastatic tumor tissues.

Topics: Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Epidermal Growth Factor; Epoetin Alfa; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Hepatocyte Growth Factor; HT29 Cells; Humans; Insulin-Like Growth Factor I; Male; Ovarian Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Receptor, IGF Type 1; Receptors, Erythropoietin; Signal Transduction

Transforming growth factor-α (TGF-α), like epidermal growth factor (EGF) and amphiregulin (AREG) binds exclusively to EGF receptor (EGFR). We have previously demonstrated that EGF, AREG and TGF-α down-regulate E-cadherin and induce ovarian cancer cell invasion, though whether these ligands use the same molecular mediators remains unknown. We now show that, like EGF, TGF-α- and AREG-induced E-cadherin down-regulation involves both EGFR and HER2. However, in contrast to EGF and AREG, the transcription factor Snail is not required for TGF-α-induced E-cadherin down-regulation. This study shows that TGF-α uses common and divergent molecular mediators to regulate E-cadherin expression and cell invasion.

Topics: Amphiregulin; Cadherins; Cell Line, Tumor; Down-Regulation; EGF Family of Proteins; Epidermal Growth Factor; Female; Humans; Neoplasm Invasiveness; Ovarian Neoplasms; Receptor, ErbB-2; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Studies have shown that TNFR1 is a key factor in the tumor microenvironment that is dependent on the TNF-α-initiated cascade for tumorigenesis. In this present study, we found that TNFR1 is over-expressed in ovarian cancer, which is relevant to both clinical survival and disease free status. Knockdown of TNFR1 dramatically attenuates malignant phenotypes, including proliferation and colony growth in soft agar, as well as glycolysis in ovarian cancer cells. Unexpectedly, knocking down TNFR1 blocks EGF-induced p-AKT and p-p70S6K expression and EGF-induced cell transformation through PIK3-p110beta rather than p110alpha expression. Taken together, our data provide evidence that TNFR1 plays a critical role in ovarian cancer and show that the EGF induced signaling pathway is independent of the TNF-α triggering cascade signal. Therefore, TNFR1 may serve as a prognostic molecule in ovarian cancer.

Topics: Animals; Cell Line, Tumor; Cell Transformation, Neoplastic; Class I Phosphatidylinositol 3-Kinases; Disease Models, Animal; Epidermal Growth Factor; Female; Gene Expression; Gene Knockdown Techniques; Glucose; Glycolysis; Heterografts; Humans; Immunohistochemistry; Lactic Acid; Mice; Ovarian Neoplasms; Phenotype; Phosphatidylinositol 3-Kinases; Prognosis; Receptors, Tumor Necrosis Factor, Type I; Signal Transduction; Tumor Burden

Time-to-event regression models are a critical tool for associating survival time outcomes with molecular data. Despite mounting evidence that genetic subgroups of the same clinical disease exist, little attention has been given to exploring how this heterogeneity affects time-to-event model building and how to accommodate it. Methods able to diagnose and model heterogeneity should be valuable additions to the biomarker discovery toolset.. We propose a mixture of survival functions that classifies subjects with similar relationships to a time-to-event response. This model incorporates multivariate regression and model selection and can be fit with an expectation maximization algorithm, we call Cox-assisted clustering. We illustrate a likely manifestation of genetic heterogeneity and demonstrate how it may affect survival models with little warning. An application to gene expression in ovarian cancer DNA repair pathways illustrates how the model may be used to learn new genetic subsets for risk stratification. We explore the implications of this model for censored observations and the effect on genomic predictors and diagnostic analysis.. R implementation of CAC using standard packages is available at https://gist.github.com/programeng/8620b85146b14b6edf8f Data used in the analysis are publicly available.

Topics: Algorithms; Cluster Analysis; DNA Repair; Epidermal Growth Factor; Epiregulin; Female; Gene Expression; Genetic Heterogeneity; Humans; Models, Genetic; Models, Statistical; Ovarian Neoplasms; Regression Analysis; Survival Analysis

Heparan sulfate (HS) is a component of cell surface and extracellular matrix proteoglycans that regulates numerous signaling pathways by binding and activating multiple growth factors and chemokines. The amount and pattern of HS sulfation are key determinants for the assembly of the trimolecular, HS-growth factor-receptor, signaling complex. Here we demonstrate that HS 6-O-sulfotransferases 1 and 2 (HS6ST-1 and HS6ST-2), which perform sulfation at 6-O position in glucosamine in HS, impact ovarian cancer angiogenesis through the HS-dependent HB-EGF/EGFR axis that subsequently modulates the expression of multiple angiogenic cytokines. Down-regulation of HS6ST-1 or HS6ST-2 in human ovarian cancer cell lines results in 30-50% reduction in glucosamine 6-O-sulfate levels in HS, impairing HB-EGF-dependent EGFR signaling and diminishing FGF2, IL-6, and IL-8 mRNA and protein levels in cancer cells. These cancer cell-related changes reduce endothelial cell signaling and tubule formation in vitro. In vivo, the development of subcutaneous tumor nodules with reduced 6-O-sulfation is significantly delayed at the initial stages of tumor establishment with further reduction in angiogenesis occurring throughout tumor growth. Our results show that in addition to the critical role that 6-O-sulfate moieties play in angiogenic cytokine activation, HS 6-O-sulfation level, determined by the expression of HS6ST isoforms in ovarian cancer cells, is a major regulator of angiogenic program in ovarian cancer cells impacting HB-EGF signaling and subsequent expression of angiogenic cytokines by cancer cells.

Topics: Animals; Cell Line, Tumor; Culture Media, Conditioned; Cytokines; Disaccharides; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glucosamine; Humans; Interleukin-6; Interleukin-8; Mice; Mice, Inbred NOD; Neoplasm Transplantation; Neovascularization, Pathologic; Ovarian Neoplasms; Platelet Endothelial Cell Adhesion Molecule-1; Signal Transduction; Sulfotransferases

Systemic side effects and low aqueous solubility have limited the clinical use of cisplatin (CDDP) in ovarian carcinoma and have contributed to failures in developing effective drug delivery systems. In order to develop a novel drug delivery system with enhanced efficacy and minimal adverse effects, we exploited the properties of sodium alginate (SA) to synthesize CDDP-SA conjugate (CS), which is highly soluble and readily incorporated into liposomes (CS-PEG-Lip). Epidermal growth factor receptor (EGFR) is overexpressed in many ovarian cancers, therefore we modified EGF on the liposomes (CS-EGF-Lip) to specifically target EGFR-expressing tumors, thereby increasing the bioavailability and efficacy of CDDP. In vitro experiments confirmed that EGF-Lip selectively recognized EGFR-positive SKOV3 cells and effectively penetrated tumor spheroids. We demonstrated that CS-EGF-Lip possessed satisfactory size distribution and exhibited significantly improved encapsulation and loading efficiency. Furthermore, CS-EGF-Lip sustained release of CDDP in vitro, suggesting that CS-EGF-Lip may retain the antitumor activity of CDDP. Inhibition of proliferation and migration was also greater with CS-EGF-Lip compared to CDDP. In vivo xenograft experiments revealed that administration of CS-EGF-Lip enhanced delivery of CDDP into ovarian tumor tissues and improved the antitumor efficacy of CDDP, while reducing nephrotoxicity and body weight loss in mice. These results suggest that CS-EGF-Lip may offer a promising strategy for CDDP delivery in the treatment of EGFR-positive ovarian carcinoma or similar tumors, with enhanced efficacy and fewer adverse effects.

Topics: Alginates; Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cisplatin; Delayed-Action Preparations; Drug Delivery Systems; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Female; Glucuronic Acid; Hexuronic Acids; Humans; Kinetics; Liposomes; Mice; Mice, Nude; Nanoparticles; Ovarian Neoplasms; Polyethylene Glycols; Rhodamines; Spheroids, Cellular

Elevated expression of cyclooxygenase 2 (COX2 (PTGS2)) has been reported to occur in human ovarian cancer and to be associated with poor prognosis. We have previously demonstrated that COX2-derived prostaglandin E2 (PGE2) promotes human ovarian cancer cell invasion. We had also demonstrated that epidermal growth factor (EGF) induces human ovarian cancer cell invasion by downregulating the expression of E-cadherin through various signaling pathways. However, it remains unclear whether COX2 and PGE2 are involved in the EGF-induced downregulation of E-cadherin expression and cell invasion in human ovarian cancer cells. In this study, we showed that EGF treatment induces COX2 expression and PGE2 production in SKOV3 and OVCAR5 human ovarian cancer cell lines. Interestingly, COX2 is not required for the EGF-induced downregulation of E-cadherin expression. In addition, EGF treatment activates the phosphatidylinositol-3-kinase (PI3K)/Akt and cAMP response element-binding protein (CREB) signaling pathways, while only the PI3K/Akt pathway is involved in EGF-induced COX2 expression. Moreover, we also showed that EGF-induced cell invasion is attenuated by treatment with a selective COX2 inhibitor, NS-398, as well as PGE2 siRNA. This study demonstrates an important role for COX2 and its derivative, PGE2, in the mediation of the effects of EGF on human ovarian cancer cell invasion.

Topics: Cadherins; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Down-Regulation; Epidermal Growth Factor; Female; Humans; Neoplasm Invasiveness; Nitrobenzenes; Ovarian Neoplasms; RNA, Small Interfering; Sulfonamides

Aberrant epidermal growth factor receptor (EGFR) activation is associated with ovarian cancer progression. In this study, we report that the EGFR ligand amphiregulin (AREG) stimulates cell invasion and down-regulates E-cadherin expression in two human ovarian cancer cell lines, SKOV3 and OVCAR5. In addition, AREG increases the expression of transcriptional repressors of E-cadherin including SNAIL, SLUG and ZEB1. siRNA targeting SNAIL or SLUG abolishes AREG-induced cell invasion. Moreover, ERK1/2 and AKT pathways are involved in AREG-induced E-cadherin down-regulation and cell invasion. Finally, we show that three EGFR ligands, AREG, epidermal growth factor (EGF) and transforming growth factor-α (TGF-α), exhibit comparable effects in down-regulating E-cadherin and promoting cell invasion. This study demonstrates that AREG induces ovarian cancer cell invasion by down-regulating E-cadherin expression.

Topics: Amphiregulin; Cadherins; Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; Female; Gene Knockdown Techniques; Humans; MAP Kinase Signaling System; Neoplasm Invasiveness; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor alpha

Construction on the nanoparticles with lower toxicity and specific tumor targeting properties is challenging and requires careful design of composition, size, physicochemical properties tailored for the nanoparticles. Here the epidermal growth factor (EGF) modified methoxy polyethylene glycol-polylactic-co-glycolic acid-polylysine (mPEG-PLGA-PLL) encapsulated cisplatin (CDDP) nanoparticles (CDDP-NPs-EGF) was prepared to for solving the toxicity of CDDP and improving therapeutic efficiency. The remarkable features of CDDP-NPs-EGF are increasing cytotoxicity that attribute to effective cell cycle arrest and high cell apoptosis in vitro. In vivo, the CDDP-NPs-EGF change drug distribution, decrease the nephrotoxicity of CDDP and improve significantly therapeutic efficiency without inducing obvious system toxicity, verifying its key role of the CDDP-NPs-EGF in lowering drug toxicity and enhancing the antitumor efficiency for SKOV3 cancer in mice.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Survival; Cisplatin; Epidermal Growth Factor; Female; Flow Cytometry; Humans; Mice; Microscopy, Confocal; Nanoparticles; Ovarian Neoplasms; Polyesters; Polyethylene Glycols; Polylysine; Tissue Distribution; Treatment Outcome; Xenograft Model Antitumor Assays

Overexpression of HER2 is correlated with a poor prognosis in many types of human cancers. Due to the interaction between HER2 and other ErbB receptors, HER2 is implicated in the EGF family of ligands-regulated tumor progression. In ovarian cancer, although the relationships between HER2 amplification and patient prognosis remain controversial, the underlying molecular mechanisms of HER2-mediated tumor progression are not fully understood. Our previous studies demonstrated that EGF induces ovarian cancer cell invasion by down-regulating E-cadherin expression through the up-regulation of its transcriptional repressors, Snail and Slug. It has been shown that overexpression of HER2 down-regulates E-cadherin expression in human mammary epithelial cells. However, whether HER2 mediates EGF-induced down-regulation of E-cadherin remains unknown. In this study, we examined the potential role of HER2 in EGF-induced down-regulation of E-cadherin and increased cell invasion. We show that EGF treatment induces the interaction of EGFR with HER2 and increases the activation of HER2 in human ovarian cancer cells; we also show that these effects are diminished by knockdown of EGFR. Importantly, treatment with HER2-specific tyrosine kinase inhibitor, AG825, and HER2 siRNA diminished the up-regulation of Snail and Slug as well as the down-regulation of E-cadherin by EGF. Finally, we also show that EGF-induced cell invasion was attenuated by treatment with HER2 siRNA. This study demonstrates an important role for HER2 in mediating the effects of EGF on Snail, Slug and E-cadherin expression as well as invasiveness in human ovarian cancer cells.

Topics: Benzothiazoles; Cadherins; Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; Female; Humans; Neoplasm Invasiveness; Ovarian Neoplasms; Receptor, ErbB-2; Snail Family Transcription Factors; Transcription Factors; Tyrphostins; Up-Regulation

Loss of the cell adhesion protein E-cadherin increases the invasive capability of ovarian cancer cells. We have previously shown that epidermal growth factor (EGF) downregulates E-cadherin and induces ovarian cancer cell invasion through the H(2)O(2)/p38 MAPK-mediated upregulation of the E-cadherin transcriptional repressor Snail. However, the molecular mechanisms underlying the EGF-induced downregulation of E-cadherin are not fully understood. In the current study, we demonstrated that treatment of two ovarian cancer cell lines, SKOV3 and OVCAR5, with EGF induced the expression of the transcription factor Egr-1, and this induction was abolished by small interfering RNA (siRNA)-mediated depletion of the EGF receptor. EGF-induced Egr-1 expression required the activation of the ERK1/2 and PI3K/Akt signaling pathways and was unrelated to EGF-induced H(2)O(2) production and activation of the p38 MAPK pathway. Moreover, depletion of Egr-1 with siRNA abolished the EGF-induced downregulation of E-cadherin and increased cell invasion. Interestingly, siRNA depletion of Egr-1 attenuated the EGF-induced expression of Slug, but not that of Snail. Moreover, chromatin immunoprecipitation (ChIP) analysis showed that Slug is a target gene of Egr-1. These results provide evidence that Egr-1 is a mediator that is involved in the EGF-induced downregulation of E-cadherin and increased cell invasion. Our results also demonstrate that EGF activates two independent signaling pathways, which are the H(2)O(2)/p38 MAPK-mediated upregulation of Snail expression and the Egr-1-mediated upregulation of Slug expression. These two signaling pathways contribute to the EGF-induced downregulation of E-cadherin, which subsequently increases the invasive capability of ovarian cancer cells.

Topics: Cadherins; Cell Line, Tumor; Down-Regulation; Early Growth Response Protein 1; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Ovarian Neoplasms; RNA, Small Interfering; Snail Family Transcription Factors; Transcription Factors; Transfection

Hypoxia-inducible factor 1α (HIF-1α) regulates the transcription of a number of genes under hypoxia and other extracellular stimulations. It has been shown that E-cadherin is down-regulated by epidermal growth factor receptor (EGF) stimulation, and that cells with low E-cadherin expression are more invasive. Our recent study demonstrated a novel mechanism by which EGF down-regulates E-cadherin expression through production of hydrogen peroxide (H(2)O(2)) and the activation of p38 MAPK in human ovarian cancer cells. In this study, we were interested in examining the potential role of HIF-1α in cell invasion under normoxic conditions, specifically when cells are treated with EGF, which is known to down-regulate E-cadherin and increase invasiveness. We show that EGF treatment induces HIF-1α expression in two human ovarian cancer cell lines (SKOV3 and OVCAR5), and that this effect is diminished by treatment with a membrane-permeable H(2)O(2) scavenger, PEG-catalase. However, the induction of HIF-1α by EGF did not require the activation of p38 MAPK. Treatment with siRNA targeting HIF-1α reduces both basal and EGF-induced HIF-1α levels. Importantly, treatment with HIF-1α siRNA diminishes the up-regulation of Snail and Slug as well as the down-regulation of E-cadherin by EGF. The involvement of HIF-1α in the down-regulation of E-cadherin was confirmed with cobalt chloride (CoCl(2)), a hypoxia-mimetic reagent. Finally, we also show that EGF-induced cell invasion is attenuated by treatment with HIF-1α siRNA. This study demonstrates an important role for HIF-1α in mediating the effects of EGF on Snail, Slug and E-cadherin expression as well as invasiveness in human ovarian cancer cells.

Topics: Cadherins; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cobalt; Down-Regulation; Epidermal Growth Factor; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Hydrogen Peroxide; Hypoxia-Inducible Factor 1, alpha Subunit; Ovarian Neoplasms; Snail Family Transcription Factors; Transcription Factors

While ovarian cancer remains the most lethal gynecological malignancy in the United States, there are no biomarkers available that are able to predict therapeutic responses to ovarian malignancies. One major hurdle in the identification of useful biomarkers has been the ability to obtain enough ovarian cancer cells from primary tissues diagnosed in the early stages of serous carcinomas, the most deadly subtype of ovarian tumor. In order to detect ovarian cancer in a state of hyperproliferation, we analyzed the implications of molecular signaling cascades in the ovarian cancer cell line OVCAR3 in a temporal manner, using a mass-spectrometry-based proteomics approach. OVCAR3 cells were treated with EGF(1), and the time course of cell progression was monitored based on Akt phosphorylation and growth dynamics. EGF-stimulated Akt phosphorylation was detected at 12 h post-treatment, but an effect on proliferation was not observed until 48 h post-exposure. Growth-stimulated cellular lysates were analyzed for protein profiles between treatment groups and across time points using iTRAQ labeling and mass spectrometry. The protein response to EGF treatment was identified via iTRAQ analysis in EGF-stimulated lysates relative to vehicle-treated specimens across the treatment time course. Validation studies were performed on one of the differentially regulated proteins, lysosomal-associated membrane protein 1 (LAMP-1), in human tissue lysates and ovarian tumor tissue sections. Further, tissue microarray analysis was performed to demarcate LAMP-1 expression across different stages of epithelial ovarian cancers. These data support the use of this approach for the efficient identification of tissue-based markers in tumor development related to specific signaling pathways. LAMP-1 is a promising biomarker for studies of the progression of EGF-stimulated ovarian cancers and might be useful in predicting treatment responses involving tyrosine kinase inhibitors or EGF receptor monoclonal antibodies.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Cystadenoma, Serous; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Lysosomal Membrane Proteins; Neoplasm Grading; Ovarian Neoplasms; Peptides; Phosphatidylinositol 3-Kinases; Phosphorylation; Proteomics; Proto-Oncogene Proteins c-akt; Signal Transduction; Tandem Mass Spectrometry; Tissue Array Analysis

The aim of this study was to better understand the mechanisms of tumor development and disease progression in human epithelial ovarian cancer. Fifty genes were screened for gene signature; 20 expressed genes were assessed in tumor and normal samples of EOC patients by RT-PCR. Expression of UBE2I, EGF, TAL2 and ILF3 was validated by qPCR on the ABI Prism 7000 Detection System. ERCC1 and XPB expression was previously determined by RT-PCR in these specimens. Statistical analyses include two-sided Kruskal-Wallis test, pairwise comparison, Pearson correlation coefficient and paired t-test. In comparison to normal samples, 6 genes demonstrated distinct expression patterns in tumor tissues, with high expression observed for ERCC1, XPB and ILF3 (p=0.001, 0.0007 and 0.002, respectively) and low expression observed for TAL2 and EGF (both p<0.0001). This differential expression pattern between normal and tumor tissues may reflect in part the development of ovarian cancer. Significant differences in expression patterns of these genes in clear cell, endometrioid, mucinous and serous ovarian cancer were observed. Comparison of expression of any two EOC subtypes revealed multiple gene involvement in histopathological differentiation and cancer progression. A positive association was found between ERCC1 and XPB expression (r=0.53, p<0.0001) and between TAL2 and EGF expression (r=0.817, p<0.0001) suggesting the existence of gene linkage in these tumors. The differences in expression patterns of studied genes between tumors and normal specimens, between histological subtypes and correlations among studied genes, may indicate their involvement in tumor growth and disease progression in human epithelial ovarian cancer. Further investigation of these genes may enable better understanding of the molecular mechanism of tumorigenesis and identification of potential biomarkers.

Topics: Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Carcinoma, Ovarian Epithelial; DNA Helicases; DNA-Binding Proteins; Endonucleases; Epidermal Growth Factor; Female; Gene Expression Profiling; Humans; Neoplasm Proteins; Neoplasms, Glandular and Epithelial; Nuclear Factor 90 Proteins; Ovarian Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Ubiquitin-Conjugating Enzymes

Targeted photosensitizer delivery to EGFR-expressing cells was achieved in the present study using a high purity, targeted photoimmunoconjugate (PIC). When the PDT agent, benzoporphyrin derivative monoacid ring A (BPD) was coupled to an EGFR-targeting antibody (cetuximab), we observed altered cellular localization and selective phototoxicity of EGFR-positive cells, but no phototoxicity of EGFR-negative cells. Cetuximab in the PIC formulation blocked EGF-induced activation of the EGFR and downstream signaling pathways. Our results suggest that photoimmunotargeting is a useful dual strategy for the selective destruction of cancer cells and also exerts the receptor-blocking biological function of the antibody.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cetuximab; Dermatitis, Phototoxic; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Photochemotherapy; Photosensitizing Agents; Porphyrins; Signal Transduction; Verteporfin

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

Topics: Antigens, CD; Antigens, Neoplasm; Antineoplastic Agents; Cell Adhesion Molecules; Cell Line, Tumor; Cell Movement; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Intercellular Junctions; MAP Kinase Signaling System; Mutation; Neoplasm Proteins; Ovarian Neoplasms; Pseudopodia; RNA, Messenger; RNA, Neoplasm; Up-Regulation

Integrins function as cell-extracellular matrix adhesion proteins and have been implicated in tumor progression. In ovarian tumors, elevated integrin β1 expression correlates with high clinical stage and poor patient survival. In this study, we report that EGF treatment up-regulated integrin β1 mRNA and protein levels in ovarian cancer cells. Moreover, pharmacological inhibition of MEK totally abolished EGF-induced integrin β1 up-regulation and cell invasion suggesting that MAPK/ERK signaling is required for EGF-induced integrin β1 up-regulation and cell invasion. Furthermore, we found that knockdown of integrin β1 expression reduced the intrinsic invasiveness of ovarian cancer cells and the EGF-induced cell invasion. Finally, we found that overexpression of integrin β1 was sufficient to promote ovarian cancer cell invasion. This study demonstrates that integrin β1 mediates EGF-induced cell invasion in ovarian cancer.

Topics: Cell Line, Tumor; Epidermal Growth Factor; Female; Gene Knockdown Techniques; Humans; Integrin beta1; MAP Kinase Signaling System; Neoplasm Invasiveness; Ovarian Neoplasms; Up-Regulation

Ovarian cancer progression is correlated with accumulation of aberrant CpG island methylation. In ovarian cancer, ascites fluid contains numerous Epidermal-Growth-Factor-Receptor (EGFR) activators, which could result in a tumor microenvironment of constant EGFR activation. Signaling pathways downstream of EGFR, such as Ras, regulate DNA methylation. We hypothesized that chronic EGFR activation could alter DNA methylation. We found that EGFR activation increased DNA methyltransferase (DNMT) activity acutely, as well as after long-term EGF treatment or expression of a mutationally activated EGFR. Furthermore, this increase in DNMT activity was dependent on EGFR catalytic activity and resulted in increased global DNA methylation. Additionally, treatment with the DNMT inhibitor/hypomethylating agent 5-Aza-2'-deoxycytidine (AZA) inhibited the EGF induced increase of both DNMT activity and global methylation. These data support a role for EGFR in the process of accumulated DNA methylation during ovarian cancer progression and suggest that epigenetic therapy may be beneficial for the treatment of ovarian cancer.

Topics: Cell Line; Cell Line, Tumor; DNA Methylation; DNA Modification Methylases; Enzyme Activation; Epidermal Growth Factor; Epigenesis, Genetic; ErbB Receptors; Female; Humans; Ovarian Neoplasms

We have shown that ezrin expression correlates with ovarian epithelial cancer (OVCA) cell proliferation and metastatic behavior. In this study, we evaluated ezrin expression in transformed ovarian superficial epithelial cells (OSE) in ovarian clefts and in culture.. Immunohistochemistry and Western blotting for immunoreactive ezrin (ir-ezrin) in normal ovarian tissue, cultured OSE, and ovarian epithelial cancer cells.. While ir-ezrin was not demonstrable in normal cuboidal surface cells or interior ovarian organelles, cells lining the ovarian clefts strongly expressed ir-ezrin. Long-term culture of OSE increased ezrin expression and cytological abnormalities. Administration of estradiol and insulin at levels reported in inclusions dramatically induced OSE ir-ezrin expression to OVCA levels and membrane specializations; ruffling, pseudopodia and filopodia. Moreover epidermal growth factor (EGF) drastically increased ezrin translocation in OSE cells in a time-dependent manner.. Ezrin expression by OSE increases during transformation. Ezrin expression is responsive to estradiol and growth factors previously shown to be present in ovarian inclusions. These findings suggest that the microenvironment in ovarian inclusions and clefts contributes to the development of OVCA. Our findings elaborate on the mechanism of the ovarian origin of OVCA.

Topics: Blotting, Western; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Transformation, Neoplastic; Cells, Cultured; Cystadenocarcinoma, Serous; Cytoskeletal Proteins; Epidermal Growth Factor; Epithelial Cells; Estradiol; Female; Gene Expression; Humans; Immunohistochemistry; Insulin; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Ovary

In high-grade ovarian cancer cultures, it has been shown that epidermal growth factor (EGF) induces cell invasion by activating an epithelial-mesenchymal transition (EMT). However, the effect of EGF on serous borderline ovarian tumors (SBOT) and low-grade serous carcinomas (LGC) cell invasion remains unknown. Here, we show that EGF receptor (EGFR) was expressed, that EGF treatment increased cell migration and invasion in two cultured SBOT cell lines, SBOT3.1 and SV40 large T antigen-infected SBOT cells (SBOT4-LT), and in two cultured LGC cell lines, MPSC1 and SV40 LT/ST-immortalized LGC cells (ILGC). However, EGF induced down-regulation of E-cadherin and concurrent up-regulation of N-cadherin in SBOT cells but not in LGC cells. In SBOT cells, the expression of the transcriptional repressors of E-cadherin, Snail, Slug and ZEB1 were increased by EGF treatment. Treatment with EGF led to the activation of the downstream ERK1/2 and PI3K/Akt. The MEK1 inhibitor PD98059 diminished the EGF-induced cadherin switch and the up-regulation of Snail, Slug and ZEB1 and the EGF-mediated increase in SBOT cell migration and invasion. The PI3K inhibitor LY294002 had similar effects, but it could not block the EGF-induced up-regulation of N-cadherin and ZEB1. This study demonstrates that EGF induces SBOT cell migration and invasion by activating EMT, which involves the activation of the ERK1/2 and PI3K/Akt pathways and, subsequently, Snail, Slug and ZEB1 expression. Moreover, our results suggest that there are EMT-independent mechanisms that mediate the EGF-induced LGC cell migration and invasion.

Topics: Cadherins; Cell Line, Tumor; Cell Movement; Chromones; Cystadenocarcinoma, Serous; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Female; Flavonoids; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Morpholines; Neoplasm Invasiveness; Ovarian Neoplasms

Ovarian adenocarcinomas, like human ovarian surface epithelial cells, form functional tight junctions. Tight junction molecules claudin-3 and claudin-4, which are the receptors of Clostridium perfringens enterotoxin (CPE), are abnormally upregulated in epithelial ovarian cancers of all subtypes including, mucinous cystadenocarcinoma and serous cystadenocarcinoma. Clostridium perfringens enterotoxin may be a novel tumor-targeted therapy for ovarian cancers. In epithelial ovarian cancers, overexpression of epidermal growth factor receptor has been observed and the exogenous ligand EGF induces epithelial-mesenchymal transition in ovarian surface epithelium. Epidermal growth factor (EGF) signaling modulates expression of claudins with changes of fence and barrier functions in various cell types. However, the regulation of tight junctions by EGF in ovarian cancers remains unclear. In the present study, to investigate the mechanisms of the regulation of tight junctions in ovarian cancers, ovarian cancer cell lines mucinous cystadenocarcinoma (MCAS) and serous cystadenocarcinoma (HUOA) were treated with EGF. Epidermal growth factor downregulated claudin-3 in MCAS and claudin-4 in HUOA by inducing degradation of the proteins with changes in structures and functions of tight junctions via the MEK/ERK or PI3K/Akt signaling pathway. In addition, in HUOA but not MCAS, EGF downregulated the cytotoxic effect of CPE via claudin-4. Thus, there were different mechanisms for regulation of claudins by EGF between subtypes of epithelial ovarian cancer cells in vitro. These results indicate that EGF may affect claudins and tight junctional functions in ovarian cancer cells during cancer progression.

Topics: Cell Line, Tumor; Claudin-1; Claudin-3; Claudin-4; Claudins; Cystadenocarcinoma, Serous; Epidermal Growth Factor; Female; Humans; Ovarian Neoplasms; RNA, Messenger; Signal Transduction; Tight Junctions

Janus-activated kinase-2 (JAK2) plays an important role in the activation of signal transducer and activation of transcription 3 (STAT3), which is over expressed in majority of ovarian tumors. We have reported previously that diindolylmethane (DIM) induces apoptosis in ovarian cancer cells by inhibiting STAT3. However, the role of JAK2 in our model was not yet understood and hence evaluated in this report. SKOV-3 human ovarian cancer cells were used to evaluate concentration and time dependent effects of DIM. Interleukin 3 (IL-3) and epidermal growth factor (EGF) were used to activate JAK2. Tumor xenograft studies were used to determine modulation of JAK2 in vivo. DIM treatment blocked the phosphorylation of JAK2 at Tyr-1007 in a concentration-dependent manner. In a time-dependent study, inhibition of JAK2 by DIM was as early as 1 h, which was followed by the inhibition of STAT3 and survivin. IL-3-induced phosphorylation of JAK2 and STAT3 was significantly blocked by DIM. IL-3 treatment blocked DIM-induced apoptosis. EGF treatment resulted in the activation of JAK2 and STAT3 but suppressed by DIM. These results indicate the involvement of cytokines and growth factors in the activation of JAK2/STAT3 and that DIM suppress their activation. Furthermore, DIM in combination with cisplatin drastically reduced the phosphorylation of JAK2 when compared to cisplatin alone. Western blot analysis of tumors from DIM treated mice showed significant inhibition of JAK2 activation as compared with controls. These findings provide a rationale for further clinical investigation of DIM for its potential use alone or in combination with chemotherapy of ovarian cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cisplatin; Dose-Response Relationship, Drug; Drug Interactions; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Female; Humans; Indoles; Inhibitor of Apoptosis Proteins; Interleukin-3; Janus Kinase 2; Mice; Mice, Nude; Ovarian Neoplasms; Phosphorylation; Signal Transduction; STAT3 Transcription Factor; Survivin

This study aimed to investigate serum levels of epidermal growth factor (EGF), transforming growth factor α (TGF-α), and c-erbB2 in patients with ovarian cancer.. In this retrospective cohort study, the study and control groups were composed of 43 women with a prediagnosis of ovarian cancer and 43 healthy women, respectively. Blood samples from all women were obtained and studied by enzyme-linked immunosorbent assay kits for EGF, TGF-α, and c-erbB2. After surgery of the study group, ovarian cancer was confirmed and compared with control group. Stage, grade, and histological types were defined after histopathologic examination, and subgroups were constructed and compared.. Serum EGF, TGF-α, and c-erbB2 levels were significantly increased in study group compared with those in the control group (P < 0.001). There were no differences in serum levels of EGF, TGF-α, and c-erbB2 among all stages, grades, and histological types of ovarian cancer. If 47.90 pg/mL was selected as the cutoff value, EGF has an 80% sensitivity and a 65% specificity for detecting ovarian cancer. The cutoff value of 41,095.00 pg/mL for TGF-α has a 90% sensitivity and a 72% specificity for detecting ovarian cancer. The c-erbB2 level of 4.63 pg/mL as the cutoff value has an 83% sensitivity and a 76% specificity for predicting ovarian cancer.. Serum levels of EGF, TGF-α, and c-erbB2 may be used for diagnosing ovarian cancer.

Topics: Adenocarcinoma, Clear Cell; Adenocarcinoma, Mucinous; Biomarkers, Tumor; Case-Control Studies; Cystadenocarcinoma, Serous; Endometrial Neoplasms; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Humans; Middle Aged; Neoplasm Grading; Neoplasm Staging; Ovarian Neoplasms; Prognosis; Receptor, ErbB-2; Retrospective Studies; ROC Curve; Transforming Growth Factor alpha

This study was performed to develop a functional poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG)-bearing amino-active end group for peptide conjugation.. PLGA was preactivated following by copolymerization with PEG diamine. The resulting amphiphilic PLGA-PEG copolymer bearing 97.0% of amino end groups had a critical micelle concentration of 3.0 × 10⁻⁸ mol/L, and the half-effective inhibition concentration (IC₅₀) of the prepared PLGA-PEG nanoparticles was >100 mg/mL, which was much higher than that of PLGA nanoparticles (1.02 ± 0.37 mg/mL). The amphiphilic properties of PLGA-PEG spontaneously formed a core-shell conformation in the aqueous environment, and this special feature provided the amino group on the PEG chain scattered on the surface of PLGA-PEG nanoparticles for efficient peptide conjugation. The peptide-conjugated PLGA-PEG nanoparticles showed three-fold higher uptake than peptide-free PLGA-PEG nanoparticles in a SKOV3 cell line with high expression of epidermal growth factor receptor. Both peptide-conjugated and peptide-free PLGA-PEG nanoparticles were used as nanocarriers for delivery of doxorubicin. Although the rate of release of doxorubicin from both nanoparticles was similar, drug release at pH 4.0 (500 U lipase) was faster than at pH 7.4. The IC₅₀ of doxorubicin-loaded peptide-conjugated PLGA-PEG nanoparticles in SKOV3 cells (0.05 ± 0.03 μg/mL) was much lower (by 62.4-fold) than that of peptide-free PLGA-PEG nanoparticles (3.12 ± 1.44 μg/mL).. This in vivo biodistribution study in SKOV3 tumor-bearing mice was further promising in that accumulation of doxorubicin in tumor tissue was in the order of peptide-conjugated PLGA-PEG nanoparticles > peptide-free PLGA-PEG nanoparticles > doxorubicin solution.

Topics: Animals; Antibiotics, Antineoplastic; Cell Line, Tumor; Doxorubicin; Epidermal Growth Factor; Female; Ligands; Metabolic Clearance Rate; Mice; Mice, Nude; Nanocapsules; Organ Specificity; Ovarian Neoplasms; Peptides; Polyethylene Glycols; Polyglactin 910; Tissue Distribution

Platelet derived growth factor (PDGF) regulates gene transcription by binding to specific receptors. PDGF plays a critical role in oncogenesis in brain and other tumors, regulates angiogenesis, and remodels the stroma in physiologic conditions. Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells. The two PDGF ligands AA and BB affect expression of several miRs in ligand-specific manner; the most robust changes consisting of let-7d repression by PDGF-AA and miR-146b induction by PDGF-BB. Induction of miR-146b by PDGF-BB is modulated via MAPK-dependent induction of c-fos. We demonstrate that PDGF regulates expression of some of its known targets (e.g. cyclin D1) through miR alterations and identify the epidermal growth factor receptor (EGFR) as a new PDGF-BB target. We show that its expression and function are repressed by PDGF-induced miR-146b and that mir-146b and EGFR correlate inversely in human glioblastomas. We propose that PDGF-regulated gene transcription involves alterations in non-coding RNAs and provide evidence for a miR-dependent feedback mechanism balancing growth factor receptor signaling in cancer cells.

Topics: Becaplermin; Cell Line; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; MicroRNAs; Ovarian Neoplasms; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Signal Transduction

1alpha,25-dihydroxyvitamin D3, 1,25(OH)(2)D(3), regulates gene expression through the vitamin D receptor. The present studies identify the epidermal growth factor receptor, EGFR, as a target gene suppressed by 1,25(OH)(2)D(3) in human ovarian cancer cells. The suppression was detected at both mRNA and protein levels in vitamin D-sensitive human ovarian cancer cells. A novel vitamin D response element was identified in intron 1 of the EGFR genome, a known hotspot for its transcriptional regulation. Chromatin immunoprecipitations and reporter gene analyses showed that the intronic DNA element bound to vitamin D receptor and a co-repressor and was functional in mediating transcriptional suppression of EGFR promoter by 1,25(OH)(2)D(3) under stable transfection conditions. Consistent with the EGFR down regulation, 1,25(OH)(2)D(3) suppressed activation of the external signal regulated kinase by epidermal growth factors. Over expression of an active EGFR in vitamin D sensitive ovarian cancer cells caused resistance to 1,25(OH)(2)D(3)-induced growth suppression and diminished the hormonal regulation of cyclin D1, cyclin E, Skp2 and p27, a group of cell cycle regulators that mediate 1,25(OH)(2)D(3)-induced cell cycle arrest at G1-S checkpoint. Taken together, our studies demonstrate that 1,25(OH)(2)D(3) suppresses the response of human ovarian cancer cells to mitogenic growth factors and couple the suppression to the cell cycle arrest at G1-S checkpoint by the hormone.

Topics: Calcitriol; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Female; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Introns; Luciferases, Firefly; Ovarian Neoplasms; Signal Transduction; Vitamin D Response Element

Epiregulin (Ep) was found to be produced in non-cancer ovarian cells in response to gonadotropin stimulation as well in ovarian cancer cells in an autonomous manner. However, there were no systematic follow-up studies of Ep expression in the development of different stages of ovarian cancer. Using specific antibodies to Ep and the indirect immunocytochemistry methods, we found that in normal ovary the staining for Ep was mainly confined to the epithelial cells, while the stromal cells were only occasionally and moderately stained. In contrast in benign serous and mucinous tumors most of the tumor cells showed a clear staining in the cytoplasm. In borderline serous and mucinous tumors the staining was much more intensive, and appear occasionally in aggregated form. In serous, mucinous and endometrioid carcinomas labeling remain high, with more frequent aggregated form. It is suggested that follow-up of the expression of Ep can serve as a reliable early indication of the development of ovarian cancer. Moreover, the cytoplasmic aggregation of Ep may suggest a specific mechanism of the release of this growth factor to the extracellular space in order to exert its autocrine and paracrine effect on the family of the EGF receptors.

Topics: Adenocarcinoma, Mucinous; Biomarkers, Tumor; Epidermal Growth Factor; Epiregulin; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Staging; Ovarian Neoplasms; Ovary

MicroRNAs (miRNAs) are small noncoding RNAs that function as master regulators of posttranscriptional gene expression with each miRNA negatively regulating hundreds of genes. Lysophosphatidic acid (LPA) is a mitogenic lipid present within the ovarian tumor microenvironment and induces LPA receptor activation and intracellular signaling cascades like ERK/MAPK, leading to enhanced cellular proliferation. Here, we show that in SKOV-3 and OVCAR-3 cells, LPA stimulation at concentrations ranging from 1 nmol/L to 20 μmol/L for 30 to 60 minutes increases miR-30c-2*, and this effect is mediated through a combination of receptors because knock down of multiple LPA receptors is required for inhibition. The epidermal growth factor and platelet-derived growth factor also increase miR-30c-2* transcript expression, suggesting a broader responsive role for miR-30c-2*. Thus, we investigated the functional role of miR-30c-2* through ectopic expression of synthetic miRNA precursors of mature miRNA or antagomir transfection and observed that microRNA-30c-2* reduces, and the antagomir enhances, cell proliferation and viability in OVCAR-3, cisplatin-insensitive SKOV-3 and chemoresistant HeyA8-MDR cells. Ectopic expression of miR-30c-2* reduces BCL9 mRNA transcript abundance and BCL9 protein. Consistent with this observation, miR-30c-2* ectopic expression also reduced BCL9 luciferase reporter gene expression. In comparison with IOSE cells, all cancer cells examined showed increased BCL9 expression, which is consistent with its role in tumor progression. Taken together, this suggest that growth factor induced proliferation mediates a neutralizing response by significantly increasing miR-30c-2* which reduces BCL9 expression and cell proliferation in SKOV-3 and OVCAR-3 cells, likely as a mechanism to regulate signal transduction downstream.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genome, Human; Humans; Lysophospholipids; MicroRNAs; Neoplasm Proteins; Ovarian Neoplasms; Platelet-Derived Growth Factor; Receptors, Lysophosphatidic Acid; Signal Transduction; Transcription Factors; Transfection

Lysophosphatidic acid (LPA), is a lipid mediator that binds to G-protein coupled receptors. Epidermal growth factor (EGF), a polypeptide growth factor, binds to the EGF receptor (EGFR), a receptor tyrosine kinase. Both LPA and EGF induce responses in tumor cells that include proliferation, migration, metastasis, and induction of angiogenesis. LPA has the potential to act as an autocrine/paracrine factor and can transactivate the EGFR. This study explores the role of phospholipase D2 (PLD2) activation in LPA production, as well as cross-talk between EGF and LPA receptors. We demonstrate that EGF and LPA both stimulate production of LPA by OVCAR3 and SKOV3 human ovarian cancer cell lines. PD158780, an EGFR-selective tyrosine kinase inhibitor, blocks LPA production in response to both EGF and LPA in OVCAR3 and SKOV3 cells. Pertussis toxin, an inhibitor of LPA receptor signaling, inhibits LPA production in response to both EGF and LPA. Similar results were observed for the LPA receptor antagonist, Ki16425. Overexpression of PLD2 increases LPA production, while knockdown of PLD2 blocks EGF-induced LPA production. A phospholipase A2 (PLA2) inhibitor also blocks LPA- and EGF-induced LPA production. These results indicate that EGF stimulates LPA production in a manner that requires PLD2, and suggest that cross-talk can occur bidirectionally between EGF and LPA receptors.

Topics: Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Genetic Vectors; Humans; Lysophospholipids; Ovarian Neoplasms; Pertussis Toxin; Phospholipase D; Protein-Tyrosine Kinases; Receptor Cross-Talk; Receptors, Lysophosphatidic Acid; Transcriptional Activation; Transfection

Previously, we showed that basal activity of nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G (PKG) signaling pathway protects against spontaneous apoptosis and confers resistance to cisplatin-induced apoptosis in human ovarian cancer cells. The present study determines whether basal PKG kinase activity regulates Src family kinase (SFK) activity and proliferation in these cells. PKG-Ialpha was identified as predominant isoform in both OV2008 (cisplatin-sensitive, wild-type p53) and A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells. In both cell lines, ODQ (inhibitor of endogenous NO-induced cGMP biosynthesis), DT-2 (highly specific inhibitor of PKG-Ialpha kinase activity), and PKG-Ialpha knockdown (using small interfering RNA) caused concentration-dependent inhibition of DNA synthesis (assessed by bromodeoxyuridine incorporation), indicating an important role of basal cGMP/PKG-Ialpha kinase activity in promoting cell proliferation. DNA synthesis in OV2008 cells was dependent on SFK activity, determined using highly selective SFK inhibitor, 4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline (SKI-1). Studies using DT-2 and PKG-Ialpha small interfering RNA revealed that SFK activity was dependent on PKG-Ialpha kinase activity. Furthermore, SFK activity contributed to endogenous tyrosine phosphorylation of PKG-Ialpha in OV2008 and A2780cp cells. In vitro coincubation of recombinant human c-Src and PKG-Ialpha resulted in c-Src-mediated tyrosine phosphorylation of PKG-Ialpha and enhanced c-Src autophosphorylation/activation, suggesting that human c-Src directly tyrosine phosphorylates PKG-Ialpha and the c-Src/PKG-Ialpha interaction enhances Src kinase activity. Epidermal growth factor-induced stimulation of SFK activity in OV2008 cells increased PKG-Ialpha kinase activity (indicated by Ser(239) phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein), which was blocked by both SKI-1 and SU6656. The data suggest an important role of Src/PKG-Ialpha interaction in promoting DNA synthesis/cell proliferation in human ovarian cancer cells. The NO/cGMP/PKG-Ialpha signaling pathway may provide a novel therapeutic target for disrupting ovarian cancer cell proliferation.

Topics: Carcinoma; Cell Line, Tumor; Cell Proliferation; CSK Tyrosine-Protein Kinase; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; DNA Replication; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Nitric Oxide; Ovarian Neoplasms; Phosphorylation; Protein-Tyrosine Kinases; RNA Interference; src-Family Kinases

Rab5a is a regulatory guanosine triphosphatase that is associated with the transport and fusion of endocytic vesicles, and participates in regulation of intracellular signaling pathways embraced by cells to adapt to the specific environment. Rab5a is also correlated with lung, stomach, and hepatocellular carcinomas. Here, we detected Rab5a in paraffin-embedded samples of 20 ovarian cysts, 20 benign cystadenomas, and 39 ovarian cancers by immunohistochemistry, and observed that Rab5a expression was significantly higher in ovarian cancer (P = 0.0001). By setting up stable HO-8910 cell lines expressing Rab5a or dominant negative Rab5a (Rab5a:S34N), we found that Rab5a overexpression enhanced the cell growth by promoting G1 into S phase. In contrast, Rab5a:S34N inhibited this process. Additionally, APPL1 (adaptor protein containing PH domain, PTB domain, and Leucine zipper motif), a downstream effector of Rab5a, was also involved in promoting HO-8910 cell cycle progress. But this function was blocked by Rab5a:S34N. Laser scanning confocal microscopy represented the colocalization of APPL1 and Rab5a in the plasmolemma, which changed with the time of epidermal growth factor (EGF) stimulation. We also found APPL1 could transfer from the membranes into the nucleus where it interacted with NuRD/MeCP1 (the nucleosome remodeling and histone deacetylase multiprotein complex). NuRD is reported to be involved in the deacetylation of histone H3 and H4 to regulate nuclear transcription. So Rab5a promoted proliferation of ovarian cancer cells, which may be associated with the APPL1-related epidermal growth factor signaling pathway.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Aged, 80 and over; Cell Cycle; Cell Proliferation; Cyclin D1; Epidermal Growth Factor; Female; Gene Expression Regulation; Humans; Middle Aged; Ovarian Neoplasms; rab5 GTP-Binding Proteins; Signal Transduction

We describe for the first time a new integral molecular pathway, linking transcription factor E2F3a to epidermal growth factor receptor (EGFR) activation in ovarian cancer cells. Investigations on the role of E2F family members in EGFR-mediated mitogenic signaling revealed that E2F3a was selectively upregulated following EGFR activation, whereas all other E2F family members remained unaffected. In contrast, EGF treatment of healthy ovarian surface epithelial and mesothelial cells yielded a selective upregulation of proliferation-promoting E2F1 and E2F2 without influencing E2F3a expression. In ovarian cancer cell lines, the extent of EGF-induced proliferative stimulus was closely related to the magnitude of E2F3a increase, and proliferation inhibition by E2F3a knockdown was not overcome by EGF exposure. Furthermore, the EGFR-E2F3a axis was found to be signal transducer and activator of transcription 1/3 dependent and the ratio of IFN-regulatory factor (IRF)-1 to IRF-2 was shown to be determinative for E2F3a control. In a pilot study on 32 primary ovarian cancer specimens, a highly significant correlation between activated EGFR and E2F3a expression was disclosed. This new integral pathway in the EGFR-driven mitogenic cell response, which through its key player E2F3a was found to be essential in triggering proliferation in ovarian cancer cells, provides new insights into EGFR signaling and could represent the basis for appealing new therapeutic approaches in ovarian cancer.

Topics: Cell Growth Processes; Cell Line, Tumor; E2F3 Transcription Factor; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Interferon Regulatory Factor-1; Interferon Regulatory Factor-2; Ovarian Neoplasms; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Transfection; Up-Regulation

In ovarian cancer, it has been shown that E-cadherin is down-regulated by epidermal growth factor (EGF) receptor (EGFR) activation, and that cells with low E-cadherin expression are particularly invasive. Although it is generally believed that reactive oxygen species play important roles in intracellular signal transduction, the role of reactive oxygen species in EGF-mediated reductions in E-cadherin remains to be elucidated. In this study, we show that EGF treatment down-regulated E-cadherin by up-regulating its transcriptional repressors, Snail and Slug, in human ovarian cancer cells. Using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester staining, we found that intracellular hydrogen peroxide (H(2)O(2)) production was increased in EGF-treated cells and could be inhibited by treatment with an EGFR inhibitor, AG1478, or an H(2)O(2) scavenger, polyethylene glycol (PEG)-catalase. In addition, PEG-catalase diminished EGF-induced p38 MAPK, but not ERK1/2 or c-Jun N-terminal kinase, phosphorylation. PEG-catalase and the p38 MAPK inhibitor SB203580 abolished EGF-induced Snail, but not Slug, expression and E-cadherin down-regulation. Furthermore, the involvement of p38 MAPK in the down-regulation of E-cadherin was confirmed using specific p38alpha MAPK small interfering RNA. Finally, we also show that EGF-induced cell invasion was abolished by treatment with PEG-catalase and SB203580, as well as p38alpha MAPK small interfering RNA, and that forced expression of E-cadherin diminished intrinsic invasiveness as well as EGF-induced cell invasion. This study demonstrates a novel mechanism in which EGF down-regulates E-cadherin expression through production of H(2)O(2), activation of p38 MAPK, and up-regulation of Snail in human ovarian cancer cells.

Topics: Blotting, Western; Cadherins; Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; Female; Humans; Hydrogen Peroxide; Ovarian Neoplasms; p38 Mitogen-Activated Protein Kinases; Polymerase Chain Reaction; RNA, Small Interfering; Snail Family Transcription Factors; Transcription Factors

The epithelial-mesenchymal transition (EMT) is associated with progression and metastasis of epithelial ovarian cancer (EOC). Snail and Slug (two members of the Snail family of transcription factors) down-regulate the expression of the adhesion molecule E-cadherin and thus function as positive regulators of EMT. Their expression is associated with a more invasive phenotype of EOC. However, how their expression in EOC cells is regulated needs to be further defined. Here, we show that transforming growth factor β (TGFβ) and epidermal growth factor (EGF) synergistically induce the expression of Slug and Snail at both mRNA and protein levels in an EOC cell line OVCA429 cells. Using specific chemical inhibitors, we demonstrate that Slug and Snail expression induced by TGFβ is mediated by TGFβ/ALK5 pathway, and EGF-induced expression of Slug and Snail is MEK1/2-dependent. Interestingly, TGFβ-induced Slug expression is also MEK1/2-dependent. Further, we demonstrate that combined TGFβ and EGF stimulation is more potent than either alone in repressing the expression of E-cadherin. Functionally, combined stimulation of TGFβ and EGF enhances the mobility of OVCA429 cells and induces the production of MMP2 by OVCA429 cells more potently than either alone. Taken together, our data demonstrate that TGFβ and EGF signaling pathways synergistically induce EMT and render EOC cells a more invasive phenotype.

Topics: Cadherins; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelium; Female; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Matrix Metalloproteinase 2; Mesoderm; Ovarian Neoplasms; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta

Peritoneal dissemination of ovarian carcinoma is mediated by epithelial-mesenchymal interconversions leading to the disruption of cell-cell contact and modulation of cell-extracellular matrix (ECM) interactions. The present study was designed to evaluate the effects of epidermal growth factor (EGF) as a modulator of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) signalling and changes in integrin expression during the process similar to EMT. A fibroblastic morphology with reduced intercellular cell contacts and increased cell motility was observed in ovarian cancer cell lines in response to EGF and was concomitant with the up regulation of EMT-associated N-cadherin and vimentin expression. These changes were accompanied by an increase in alpha2, alpha6 and beta1 integrin subunits and activation of JAK2 and STAT3 signalling which was suppressed by a specific JAK2 inhibitor. Consistent with the suppression of STAT3 activity, N-cadherin and vimentin expression were abrogated and was coherent with the loss of cell motility and the expression of alpha6 and beta1 integrin subunits. Neutralizing antibodies against alpha6 and beta1 subunits inhibited cancer cell migration. A strong correlation between the expression of N-cadherin, vimentin and JAK2/STAT3 levels were detected in high-grade ovarian tumors and was consistent with the previously reported enhanced expression of alpha6 integrin subunit in advanced tumors [Ahmed N, Riley C, Oliva K, Rice G, Quinn M. Ascites induces modulation of alpha6beta1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma. British Journal of Cancer 2005;92:1475-85]. Our data incorporating the clinical samples and the cancer cell lines is the first to demonstrate that JAK2/STAT3 pathway may be one of the downstream events in EMT-like process and alpha6beta1 integrin-mediated signalling in ovarian carcinomas.

Topics: Cadherins; Cell Line, Tumor; Cell Movement; Enzyme Activation; Epidermal Growth Factor; Female; Humans; Integrin alpha6beta1; Janus Kinase 2; Ovarian Neoplasms; Signal Transduction; STAT3 Transcription Factor; Vimentin

Epidermal growth factor receptor (EGFR) is overexpressed in ovarian carcinomas, with direct or indirect activation of EGFR able to trigger tumour growth. We demonstrate significant activation of both signal transducer and activator of transcription (STAT)3 and its upstream activator Janus kinase (JAK)2, in high-grade ovarian carcinomas compared with normal ovaries and benign tumours. The association between STAT3 activation and migratory phenotype of ovarian cancer cells was investigated by EGF-induced epithelial-mesenchymal transition (EMT) in OVCA 433 and SKOV3 ovarian cancer cell lines. Ligand activation of EGFR induced a fibroblast-like morphology and migratory phenotype, consistent with the upregulation of mesenchyme-associated N-cadherin, vimentin and nuclear translocation of beta-catenin. This occurred concomitantly with activation of the downstream JAK2/STAT3 pathway. Both cell lines expressed interleukin-6 receptor (IL-6R), and treatment with EGF within 1 h resulted in a several-fold enhancement of mRNA expression of IL-6. Consistent with that, EGF treatment of both OVCA 433 and SKOV3 cell lines resulted in enhanced IL-6 production in the serum-free medium. Exogenous addition of IL-6 to OVCA 433 cells stimulated STAT3 activation and enhanced migration. Blocking antibodies against IL-6R inhibited IL-6 production and EGF- and IL-6-induced migration. Specific inhibition of STAT3 activation by JAK2-specific inhibitor AG490 blocked STAT3 phosphorylation, cell motility, induction of N-cadherin and vimentin expression and IL6 production. These data suggest that the activated status of STAT3 in high-grade ovarian carcinomas may occur directly through activation of EGFR or IL-6R or indirectly through induction of IL-6R signalling. Such activation of STAT3 suggests a rationale for a combination of anti-STAT3 and EGFR/IL-6R therapy to suppress the peritoneal spread of ovarian cancer.

Topics: Antigens, CD; beta Catenin; Cadherins; Cell Line, Tumor; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Humans; Interleukin-6; Janus Kinase 2; Leukemia Inhibitory Factor; Mesoderm; Ovarian Neoplasms; Receptor Cross-Talk; Receptors, Interleukin-6; STAT3 Transcription Factor; Vimentin

Growth factors play an essential role in regulating cellular proliferation, and lack of control is characteristic of malignant development. The epidermal growth factor (EGF) gene codifies a growth factor that binds to the EGF receptor (EGFR), which is involved in activating pathways that promote cellular proliferation, survival, migration and differentiation. The purpose of this study was to appraise the association between EGF gene A61G polymorphism with ovarian cancer susceptibility. A total of 564 DNA samples were analysed from 175 women with ovarian cancer and 389 women without cancer, through PCR-RFLP. We found a decreased risk for developing ovarian cancer in GG carriers compared to AA carriers (OR = 0.46, CI = 0.25-0.83, P = 0.010). The seemingly protective role in GG carriers was observed in women under 53 years of age (OR = 0.38, CI = 0.16-0.86, P = 0.011) and in patients diagnosed with advanced stage disease (OR = 0.38, CI = 0.18-0.81, P = 0.012). Allelic comparison evidenced similar results, with decreased risk for G allele. We further observed a linear trend for G allele in cancer risk. Moreover, we analysed the influence of genotypes in the time to onset of the disease and observed that GG carriers had ovarian cancer later than AA carriers (P = 0.035). We hypothesize that this polymorphism confers protection for ovarian cancer development.

Topics: Case-Control Studies; Epidermal Growth Factor; Female; Gene Frequency; Genetic Predisposition to Disease; Humans; Middle Aged; Ovarian Neoplasms; Polymorphism, Single Nucleotide

IRF-1 and IRF-2 expression was determined by real-time PCR in 138 ovarian cancer samples and 30 healthy ovarian biopsies and was correlated with the expression of other relevant immunologic parameters and common clinicopathologic variables. Regulation of IRF-1 and IRF-2 was evaluated by cytokine treatment of various ovarian cancer cell lines, human peritoneal mesothelial cells and ovarian surface epithelium. IRF-1 but not IRF-2 was constitutively over-expressed in 5 of 7 ovarian cancer cell lines. Both IRFs were inducible with IFN-gamma and to a lesser extent with IL-1 or TNF-alpha, but not with IL-6. Epidermal growth factor (EGF) treatment down-regulated both IRFs. In ovarian cancer samples only IRF-1, but not IRF-2 mRNA, was up-regulated when compared with healthy ovarian tissue. IRF-1 but not IRF-2 expression was significantly associated with interferon (IFN)-gamma and forkhead box P3 (FoxP3). In univariate survival analysis, strong expression of IRF-1 and IRF-2 predicted improved disease-free survival (DFS) and overall survival (OS). In Cox regression analyses, IRF-1 retained independent prognostic significance for DFS and OS and IFN-gamma for OS. In contrast to other solid tumors, IRF-2 expression cannot be regarded as a classic oncoprotein associated with poor prognosis in ovarian cancer. Of the immunologic parameters investigated, intratumoral IRF-1 expression is the most powerful independent predictor of a favorable clinical outcome.

Topics: Aged; Base Sequence; Cell Line, Tumor; DNA Primers; Down-Regulation; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Interferon Regulatory Factor-1; Interferon Regulatory Factor-2; Middle Aged; Ovarian Neoplasms; Prognosis; RNA, Messenger; Survival Analysis

Epidermal growth factor (EGF) stimulates proliferation and migration in ovarian cancer cells, and high tumor expression of the EGF system correlates with poor prognosis. Epidermal growth factor upregulates urokinase plasminogen activator receptor (uPAR) on the cell surface via 3 distinct mechanisms: rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression and cell migration in ovarian cancer cells and further to identify the ER involved.We used 7 ovarian cancer cell lines, cell migration assay, cellular binding of (125)I-uPA, cellular degradation of (125)I-uPA/PAI-1 complex, enzyme-linked immunosorbent assay for uPAR, solid-phase enzyme immunoassay for ERalpha, and quantitative polymerase chain reaction. Estradiol attenuates the stimulatory effect of EGF on cell migration and uPAR expression. Specifically, E(2) reduces the very rapid increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation and probably represents mobilization of uPAR from detergent-resistant domains such as lipid rafts. Estradiol influenced neither the amount of uPAR mRNA nor the rate of uPAR degradation or solubilization. The nuclear ER antagonists ICI 182780 and tamoxifen, which are GPR30 agonists, as well as the specifically constructed GPR30 agonist G1, mimicked the effect of E(2) on uPAR expression and cell migration. OVCAR-3 cells express mRNA for GPR30.Estradiol attenuates EGF-induced mobilization of ligated uPAR from detergent-resistant domains and subsequent migration in ovarian cancer cells. The response to various ER ligands indicates that this effect is mediated via the membrane ER GPR30.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Estradiol; Female; Humans; Ovarian Neoplasms; Receptors, Estrogen; Receptors, G-Protein-Coupled; Receptors, Urokinase Plasminogen Activator; Up-Regulation

The epidermal growth factor receptor (EGFR) is overexpressed in ovarian carcinomas and promotes cellular responses that contribute to ovarian cancer pathobiology. In addition to modulation of mitogenic and motogenic behavior, emerging data identify EGFR activation as a novel mechanism for rapid modification of the cell surface proteome. The transmembrane collagenase membrane type 1 matrix metalloproteinase (MT1-MMP, MMP-14) is a major contributor to pericelluar proteolysis in the ovarian carcinoma microenvironment and is subjected to extensive posttranslational regulation. In the present study, the contribution of EGFR activation to control of MT1-MMP cell surface dynamics was investigated. Unstimulated ovarian cancer cells display caveolar colocalization of EGFR and MT1-MMP, whereas EGFR activation prompts internalization via distinct endocytic pathways. EGF treatment results in phosphorylation of the MT1-MMP cytoplasmic tail, and cells expressing a tyrosine mutated form of MT1-MMP (MT1-MMP-Y(573)F) exhibit defective MT1-MMP internalization. As a result of sustained cell surface MT1-MMP activity, a phenotypic epithelial-mesenchymal transition is observed, characterized by enhanced migration and collagen invasion, whereas growth within three-dimensional collagen gels is inhibited. These data support an EGFR-dependent mechanism for regulation of the transition between invasive and expansive growth of ovarian carcinoma cells via modulation of MT1-MMP cell surface dynamics.

Topics: Cell Line, Tumor; Cell Proliferation; Collagen; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Female; Flow Cytometry; Humans; Matrix Metalloproteinase 14; Microscopy, Fluorescence; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Ovarian Neoplasms; Protein Transport; Tissue Scaffolds

Vasculogenesis, or recruitment of progenitors able to differentiate into endothelial-like cells, may provide an important contribution to neovessel formation in tumors. However, the factors involved in the vasculogenic process and in particular the role of the epithelial-mesenchymal transition of tumor cells have not yet been investigated. We found a CD14(+)/KDR(+) angiogenic monocyte population in undifferentiated ovarian tumors, significantly increased in the corresponding tumor metastasis. In vitro, monocyte differentiation into CD14(+)/KDR(+) cells was induced by conditioned media from the primary ovarian tumor cells expressing a mesenchymal phenotype. In contrast, the ovarian tumor cell line SKOV3 expressing an epithelial phenotype was unable to stimulate the differentiation of monocytes into CD14(+)/KDR(+) cells. When an epithelial-mesenchymal transition was induced in SKOV3, they acquired this differentiative ability. Moreover, after mesenchymal transition pleiotrophin expression by SKOV3 was increased and conversely its blockade significantly reduced monocyte differentiation. The obtained CD14(+)/KDR(+) cell population showed the expression of endothelial markers, increased the formation of capillary-like structures by endothelial cells and promoted the migration of ovarian tumor cells in vitro. In conclusion, we showed that the epithelial-mesenchymal transition of ovarian tumor cells induced differentiation of monocytes into the pro-angiogenic CD14(+)/KDR(+) population and thus it may provide a tumor microenvironment that favours vasculogenesis and metastatization of the ovarian cancer.

Topics: Adenocarcinoma; Adult; Aged; Antigens, CD; Cell Differentiation; Cell Division; Cell Line, Tumor; Epidermal Growth Factor; Epithelial Cells; Female; Flow Cytometry; Humans; Hydrocortisone; Lipopolysaccharide Receptors; Mesoderm; Middle Aged; Monocytes; Neoplasm Metastasis; Neovascularization, Pathologic; Ovarian Diseases; Ovarian Neoplasms

GnRH-II modulates ovarian cancer cells invasion and is expressed in normal ovary and ovarian epithelial cancer cells; however, the upstream regulator(s) of GnRH-II expression in these cells remains unclear. We now demonstrate that epidermal growth factor (EGF) increases GnRH-II mRNA levels in several human ovarian carcinoma cell lines and up-regulates GnRH-II promoter activity in OVCAR-3 cells in a dose-dependent manner, whereas an EGF receptor inhibitor (AG148) abolishes EGF-induced increases in GnRH-II promoter activity and GnRH-II mRNA levels. EGF increases the phosphorylation of cAMP-responsive element-binding protein (p-CREB) and its association with the coregulator, CCAAT/enhancer binding protein beta, whereas blocking the EGF-induced ERK1/2 phosphorylation with MAPK inhibitors (PD98059/U0126) markedly reduced these effects. Moreover, depletion of CREB using small interfering RNA attenuated EGF-induced GnRH-II promoter activity. Chromatin immunoprecipitation assays demonstrated that EGF induces p-CREB binding to a cAMP responsive-element within the GnRH-II promoter, likely in association with CCAAT/enhancer binding protein beta, and mutagenesis of this cAMP responsive-element prevented EGF-induced GnRH-II promoter activity in OVCAR-3 cells. Importantly, GnRH-II acts additively with EGF to promote invasion of OVCAR-3 and CaOV-3 cells, but not SKOV-3 cells that express low levels of GnRH receptor (GnRHR). Treatment with GnRHR small interfering RNA also partially inhibited the EGF-induced invasion of OVCAR-3 and CaOV-3 cells. Furthermore, EGF treatment transiently increases GnRHR levels in OVCAR-3 and CaOV-3, which likely accentuates the effects of increase GnRH-II production on cell invasion. These results provide evidence that EGF is an upstream regulator of the autocrine actions of GnRH-II on the invasive properties of ovarian cancer cells.

Topics: CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Cyclic AMP Response Element-Binding Protein; Enzyme Activation; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Gonadotropin-Releasing Hormone; Humans; Mitogen-Activated Protein Kinases; Models, Biological; Neoplasm Invasiveness; Ovarian Neoplasms; Phosphorylation; Protein Binding; Response Elements; RNA, Messenger

Cellular response to external stimuli requires propagation of corresponding signals through molecular signaling pathways. However, signaling pathways are not isolated information highways, but rather interact in a number of ways forming sophisticated signaling networks. Since defects in signaling pathways are associated with many serious diseases, understanding of the crosstalk between them is fundamental for designing molecularly targeted therapy. Unfortunately, we still lack technology that would allow high throughput detailed measurement of activity of individual signaling molecules and their interactions. This necessitates developing methods to prioritize selection of the molecules such that measuring their activity would be most informative for understanding the crosstalk. Furthermore, absence of the reaction coefficients necessary for detailed modeling of signal propagation raises the question whether simple parameter-free models could provide useful information about such pathways.. We study the combined signaling network of three major pro-survival signaling pathways: Epidermal Growth Factor Receptor (EGFR), Insulin-like Growth Factor-1 Receptor (IGF-1R), and Insulin Receptor (IR). Our study involves static analysis and dynamic modeling of this network, as well as an experimental verification of the model by measuring the response of selected signaling molecules to differential stimulation of EGF, IGF and insulin receptors. We introduced two novel measures of the importance of a node in the context of such crosstalk. Based on these measures several molecules, namely Erk1/2, Akt1, Jnk, p70S6K, were selected for monitoring in the network simulation and for experimental studies. Our simulation method relies on the Boolean network model combined with stochastic propagation of the signal. Most (although not all) trends suggested by the simulations have been confirmed by experiments.. The simple model implemented in this paper provides a valuable first step in modeling signaling networks. However, to obtain a fully predictive model, a more detailed knowledge regarding parameters of individual interactions might be necessary.

Topics: Cell Line; Cell Survival; Computer Simulation; Epidermal Growth Factor; Female; Humans; Insulin; Models, Biological; Ovarian Neoplasms; Signal Transduction; Somatomedins

This study was designed to evaluate the expression of HER receptors as a marker of sensitivity to the humanized anti-HER2 monoclonal antibody pertuzumab in ovarian cancer cells. In a recent clinical trial, low levels of HER3 mRNA have been shown to associate with pertuzumab response when combined with gemcitabine. We sought to define how pertuzumab modulated HER expression levels in ovarian cancer using cell line models to better understand differential and dynamic receptor expression in therapeutic response. Changes in HER3 mRNA expression were also assessed in pertuzumab-treated xenografts. HER3 mRNA and, to a lesser extent, HER2, were down-regulated after stimulation both with heregulin-beta1 and epidermal growth factor in a range of ovarian cancer cell lines either growth sensitive or growth resistant to pertuzumab. Pertuzumab reversed this down-regulation and the magnitude of the reversal correlated with pertuzumab sensitivity. The change in HER3 mRNA expression correlated inversely to how much the extracellular signal-regulated kinase and phosphoinositide 3-kinase pathways were dynamically activated with stimulation. Finally, up-regulation of HER3 mRNA was found in cancer xenografts treated with pertuzumab. We conclude that HER3 mRNA is down-regulated by both heregulin-beta1 and epidermal growth factor activation. This suggests that in some tumors, low HER3 mRNA expression is driven by, or dependent on, growth factor. HER3 mRNA expression is effectively reversed in pertuzumab-sensitive tumors. These data are consistent with low HER3 mRNA identifying a pertuzumab-sensitive phenotype.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression; Humans; Mice; Neuregulin-1; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Receptor, ErbB-2; Receptor, ErbB-3; RNA, Messenger; Signal Transduction; Xenograft Model Antitumor Assays

The KCl cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. This study stresses the important role of KCC4 in tumor malignant behavior. Real-time reverse transcription-PCR on samples collected by laser microdissection and immunofluorescent stainings with different KCC isoform antibodies indicate that KCC4 is abundant in metastatic cervical and ovarian cancer tissues. Insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulate KCC4 recruitment from a presumably inactive cytoplasmic pool of endoplasmic reticulum and Golgi to plasma membrane along actin cytoskeleton that is significantly inhibited by LY294002 and wortmannin. Throughout the trafficking process, KCC4 is incorporated into lipid rafts that function as a platform for the association between KCC4 and myosin Va, an actin-dependent motor protein. KCC4 and ezrin, a membrane cytoskeleton linker, colocalize at lamellipodia of migratory cancer cells. Interference with KCC activity by either an inhibitor or a dominant-negative loss-of-function mutant profoundly suppressed the IGF-I-induced membrane trafficking of KCC4 and the structural interaction between KCC4 and ezrin near the cell surface. Endogenous cancer cell invasiveness was significantly attenuated by small interfering RNA targeting KCC4, and the residual invasiveness was much less sensitive to IGF-I or EGF stimulation. In the metastatic cancer tissues, KCC4 colocalizes with IGF-I or EGF, indicating a likely in vivo stimulation of KCC4 function by growth factors. Thus, blockade of KCC4 trafficking and surface expression may provide a potential target for the prevention of IGF-I- or EGF-dependent cancer spread.

Topics: Adult; Aged; Cell Line, Tumor; Cell Membrane; Epidermal Growth Factor; Female; Fluorescent Antibody Technique; Humans; Insulin-Like Growth Factor I; Lasers; Microdissection; Middle Aged; Molecular Motor Proteins; Neoplasm Invasiveness; Ovarian Neoplasms; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Symporters; Uterine Cervical Neoplasms

Epidermal growth factor (EGF) receptor (EGFR) is frequently elevated in epithelial ovarian cancer, and E-cadherin expression is often reduced in advanced disease. In this study, we investigated a mechanism by which EGFR activation promotes disruption of adherens junctions through induction of matrix metalloproteinase 9 (MMP-9). We show that EGFR activation down-modulates E-cadherin, and broad spectrum MMP inhibition ameliorates EGF-stimulated junctional disruption and loss of E-cadherin protein. MMP-9 involvement in EGF-dependent down-regulation of E-cadherin was determined by siRNA specifically directed against MMP-9. Furthermore, treatment with recombinant MMP-9 or transient expression of MMP-9 is sufficient to reduce E-cadherin levels in differentiated ovarian tumor cells. Stable overexpression of MMP-9 led to a loss of E-cadherin and junctional integrity, and promoted a migratory and invasive phenotype. Thus, elevated MMP-9 protein expression is sufficient for junctional disruption and loss of E-cadherin in these cells. The associations between EGFR activation, MMP-9 expression, and E-cadherin were investigated in human ovarian tumors and paired peritoneal metastases wherein immunohistochemical staining for activated (phospho) EGFR and MMP-9 colocalized with regions of reduced E-cadherin. These data suggest that regulation of MMP-9 by EGFR may represent a novel mechanism for down-modulation of E-cadherin in ovarian cancer.

Topics: Adherens Junctions; Ascitic Fluid; Blotting, Western; Cadherins; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Female; Flow Cytometry; Humans; Immunoenzyme Techniques; Immunoprecipitation; Matrix Metalloproteinase 9; Microscopy, Fluorescence; Neoplasm Invasiveness; Ovarian Neoplasms; Promoter Regions, Genetic; Recombinant Proteins; RNA, Messenger; RNA, Small Interfering; Tissue Array Analysis; Transfection

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are members of the polypeptide growth factor family. The epidermal growth factor-receptor (EGF-R) is a receptor tyrosine kinase of the ErbB family. Many types of cancer, including ovarian cancer, display enhanced EGF-R immunoreactivity on their cell surface membranes. Also, an increase in TGF-alpha synthesis and secretion usually occurs in human carcinoma cell lines. In this study, we compared the immunoreactivities of TGF-alpha and EGF-R in ovarian tumors and related immunohistochemical findings to the histological type of the tumors. Formalin-fixed, paraffin wax-embedded tissue sections from 40 patients who had serous-mucinous borderline tumor and serous-mucinous adenocarcinoma of the ovary (n=10 each) were stained with hematoxylin-eosin and labeled for binding of primary antibodies against TGF-alpha and EGF-R using an avidin-biotin-peroxidase method. A semi-quantitative grading system was used to compare immunohistochemical labeling intensities. Increased immunoreactivity of EGF-R and moderate immunoreactivity of TGF-alpha was detected in adenocarcinomas. There was no significant difference in the immunoreactivity of TGF-alpha among the histologic types of ovarian tumors. The results of this study support the hypothesis that EGF-R may be a more useful marker than TGF-alpha in epithelial ovarian tumors.

Topics: Adult; Biomarkers, Tumor; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Middle Aged; Ovarian Neoplasms; Paraffin Embedding; Protein-Tyrosine Kinases; Receptor Protein-Tyrosine Kinases; Transforming Growth Factor alpha

Adenovirus gene therapy for intraperitoneal (IP) cancer is limited in clinical trials by inefficient tumor cell transduction and development of peritoneal adhesions. We have shown previously that normal virus tropism can be ablated by physically shielding the virus surface with reactive hydrophilic polymers and that linkage of novel ligands enables virus "retargeting" through chosen receptors. To achieve tumor-selective infection, polymer-coated virus was retargeted using murine epidermal growth factor (mEGF). The resulting mEGF-polymer coated adenovirus lost its normal broad tropism and transduced cells selectively via the EGF receptor (EGFR). We assessed whether this approach could be used to target lytic "virotherapy" using wild-type adenovirus (Ad5WT) in a peritoneal xenograft model of human ovarian cancer. Oncolytic activity of Ad5WT was retained following polymer coating and mEGF-retargeting. Importantly, adhesion formation was markedly decreased compared with the unmodified virus, and no dose-limiting toxicities were observed following treatment with mEGF-retargeted polymer-coated virus. Restricting virus tropism by physical coating, coupled with tumor-selective retargeting promises to combine good anticancer efficacy with acceptable toxicity, enabling application of elevated virus doses leading to an improved therapeutic outcome.

Topics: Adenoviridae; Animals; Cell Line, Tumor; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Female; Genetic Therapy; Genetic Vectors; Humans; Mice; Oncolytic Virotherapy; Ovarian Neoplasms; Polymers; Polymethacrylic Acids

Antibodies directed against the L1 cell adhesion protein inhibit growth of SKOV3ip human ovarian cancer cells in vitro and in vivo. Responses of SKOV3ip cells in vitro to anti-L1 mAb chCE7 and Genistein were investigated. Genistein potentiated the anti-proliferative and pro-apoptotic effects of chCE7 in SKOV3ip cells. A combination of mAb chCE7 and Genistein strongly reduced the sensitivity of p44/42 (Erk1,2) kinase, Src kinase and Akt kinase to extracellular stimulation with serum, Epidermal Growth Factor and Hepatocyte Growth Factor. The observed synergy of antibodies directed against L1 with Genistein could lead to a new therapeutic option for ovarian cancer.

Topics: Antibodies, Monoclonal; Anticarcinogenic Agents; Apoptosis; Blotting, Western; Cell Proliferation; Cell Survival; DNA Primers; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Female; Genistein; Hepatocyte Growth Factor; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neural Cell Adhesion Molecule L1; Ovarian Neoplasms; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; src-Family Kinases; Tumor Cells, Cultured

Aquaporin (AQP) water channels are expressed in high-grade tumor cells of different tissue origins. Based on the involvement of AQPs in angiogenesis and cell migration as well as our previous studies which show that AQP3 is involved in human skin fibroblasts cell migration, in this study, we investigated whether AQP3 is expressed in cultured human ovarian cancer cell line CaOV3 cells, and whether AQP3 expression in these cells enhances cell migration and metastatic potential.. Cultured CaOV3 cells were treated with EGF and/or various reagents and subjected to cell migration assay by phagokinetic track mobility assay or biochemical analysis for expression or activation of proteins by SDS-PAGE/Western blot analysis.. In this study, we demonstrate that AQP3 is expressed in CaOV3 cells. EGF induces CaOV3 migration and up-regulates AQP3 expression. EGF-induced cell migration is inhibited by specific AQP3 siRNA knockdown or AQP3 water transport inhibitor CuSO4 and NiCl2. We also find that curcumin, a well known anti-ovarian cancer drug, down-regulates AQP3 expression and reduces cell migration in CaOV3, and the effects of curcumin are mediated, at least in part, by its inhibitory effects on EGFR and downstream AKT/ERK activation.. Collectively, our results provide evidence for AQP3-facilitated ovarian cancer cell migration, suggesting a novel function for AQP3 expression in high-grade tumors. The results that curcumin inhibits EGF-induced up-regulation of AQP3 and cell migration, provide a new explanation for the anticancer potential of curcumin.

Topics: Antineoplastic Agents; Aquaporin 3; Blotting, Western; Cell Line, Tumor; Cell Movement; Copper Sulfate; Curcumin; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Knockdown Techniques; Humans; Nickel; Ovarian Neoplasms; Phosphoinositide-3 Kinase Inhibitors; RNA Interference; Up-Regulation

Epidermal growth factor (EGF) promotes growth of normal ovarian surface as well as malignant ovarian epithelial cells. Further, EGF receptors are present on both normal and malignant ovarian surface epithelial cells and they are often constitutively activated in many cancers. Since telomerase confers cellular immortalization and survival through increased cellular proliferation, we sought to investigate the potential role of EGF to regulate telomerase activity in normal and ovarian cancer cells. While exogenous EGF failed to activate telomerase in normal ovarian surface epithelial cells, in cancer cells we herein report that: exogenous EGF activates telomerase activity and human telomerase reverse transcriptase gene (hTERT) transcription; EGF-induced telomerase activity is ERK 1/2-dependent; EGF targets Sp1 and c-Myc binding sites within the core region of the hTERT promoter; and proline-rich tyrosine kinase 2 (Pyk2) is a key mediator of EGF-mediated telomerase activity. Together, these data show that dysregulation of EGF signaling may promote cancer cell survival through up-regulation of telomerase activity.

Topics: Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Female; Focal Adhesion Kinase 2; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Biological; Ovarian Neoplasms; Proto-Oncogene Proteins c-myc; Sp1 Transcription Factor; Telomerase

Over-expression of EGFR, as in most cases of ovarian cancer, is associated with advanced-stage disease and poor prognosis. Activation of EGFR signaling pathway is involved in increased cell proliferation, angiogenesis, metastasis and decreased apoptosis. Tyrosine kinase activity is essential for signal transduction and receptor down-regulation. However, we found in this study that tyrosine kinase activity is not necessary in ligand-induced EGFR down-regulation in ovarian cancer cell line CaOV3 cells. EGFR tyrosine kinase inhibitors, such as PD153035, AG1478, as well as non-specific tyrosine kinase inhibitor PP2 cannot reverse EGF-induced down-regulation of EGFR. These findings thus permit us to develop the following exciting but unconventional strategy to sensitize cancer cells, namely, by priming ovarian cancer cells with EGF and EGFR inhibitor PD153035, before chemotherapy. This priming procedure down-regulates EGFR without induction of mitogenic signals such as ERK and PI3K/AKT. EGF plus EGFR inhibitor-primed ovarian cancer cells display increased sensitivity to taxol-induced cell death, resistant to EGF-induced cell migration and cell proliferation as well as ERK and PI3K/AKT activation. Further studies showed that PD153035, which does not reverse ligand-induced EGFR down-regulation, blocks EGF-induced EGFR activation as well as EGFR's binding to c-cbl and Grb2. Taken together, we contend that priming with EGFR inhibitors plus EGF inhibits cell signaling pathways leading to cell proliferation and survival, while down-regulating EGFR. This priming approach sensitizes ovarian cancer cells and would ultimately result in better chemotherapeutical outcome.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Female; Mice; Ovarian Neoplasms; Paclitaxel; Protein Kinase Inhibitors; Quinazolines; Signal Transduction

Elevated expression or activity of the epidermal growth factor (EGF) receptor is common in ovarian cancer and is associated with poor patient prognosis. Our previous studies demonstrated that expression of the constitutively active mutant form of the EGF receptor (EGFRvIII) in ovarian cancer cells led to reduction in integrin alpha2 surface expression, defects in cell spreading, and disruption of focal adhesions. Inhibition of EGFRvIII catalytic activity reversed the response, suggesting that EGF receptor activation regulates integrin alpha2. In this study we found that EGF treatment resulted in a transient loss of integrin alpha2 from the cell surface. Before EGF stimulation, integrin alpha2 and EGF receptors were associated based on biochemical and immuno-colocalization approaches. After EGF treatment, EGF receptor and integrin alpha2 were internalized and segregated into different compartments. Integrin alpha2, but not EGF receptor, was associated with caveolin-1 and GM1 (Gal_1,3GalNAc_1,4(Neu5Ac-_ 2,3)Gal_1,4Glc_1,1-ceramide) gangliosides, suggesting caveolae-mediated endocytosis. Moreover, integrin alpha2 was subsequently targeted to the Golgi apparatus and the endoplasmic reticulum. Together, these findings demonstrate that activated EGF receptor transiently modulates integrin alpha2 cell surface expression and stimulates integrin alpha2 trafficking via caveolae/raft-mediated endocytosis, representing a novel mechanism by which the EGF receptor may regulate integrin-mediated cell behavior.

Topics: Caveolae; Cell Line, Tumor; Endocytosis; Endoplasmic Reticulum; Epidermal Growth Factor; ErbB Receptors; Female; Golgi Apparatus; Humans; Integrin alpha2; Ovarian Neoplasms; Protein Binding; Protein Transport

The transcription factor ZNF217 is often amplified in ovarian cancer, but its role in neoplastic progression is unknown. We introduced ZNF217-HA by adenoviral and retroviral infection into normal human ovarian surface epithelial cells (OSE), i.e., the source of ovarian cancer, and into SV40 Tag/tag expressing, p53/pRB-deficient OSE with extended but finite life spans (IOSE). In OSE, ZNF217-HA reduced cell-substratum adhesion and accelerated loss of senescent cells, but caused no obvious proneoplastic changes. In contrast, ZNF217-HA transduction into IOSE yielded two permanent lines, I-80RZ and I-144RZ, which exhibited telomerase activity, stable telomere lengths, anchorage independence and reduced serum dependence, but were not tumorigenic in SCID mice. This immortalization required short-term EGF treatment near the time of crisis. The permanent lines were EGF-independent, but ZNF217-dependent since siRNA to ZNF217 inhibited anchorage independence and arrested growth. Array CGH revealed genomic changes resembling those of ovarian carcinomas, such as amplicons at 3q and 20q, and deletions at 4q and 18, associated with underexpressed annexin A10, N-cadherin, desmocollin 3 and PAI-2, which have been reported as tumor suppressors. The lines overexpressed EEF1A2, SMARA3 and STAT1 and underexpressed other oncogenes, tumor suppressors and extracellular matrix/adhesion genes. The results implicate ZNF217 as an ovarian oncogene, which is detrimental to senescing normal OSE cells but contributes to neoplastic progression in OSE with inactivated p53/RB. The resemblance of the genomic changes in the ZNF217-overexpressing lines to ovarian carcinomas provides a unique model to investigate interrelationships between these changes and ovarian neoplastic phenotypes.

Topics: Adenoviridae; Cells, Cultured; Disease Progression; Epidermal Growth Factor; Female; Humans; Hydrocortisone; Nucleic Acid Hybridization; Ovarian Neoplasms; Phenotype; Trans-Activators

Topics: Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Multivariate Analysis; Mutation; Ovarian Neoplasms; Ovary; Prognosis; Prospective Studies; Receptor, ErbB-2; Tumor Suppressor Protein p53

Elevated expression of the epidermal growth factor (EGF) receptor (EGFR) is detected in human ovarian tumors and is associated with decreased recurrence-free and overall survival. EGFR activation affects tumor progression in part by promoting tumor invasion through the induction of prometastatic matrix metalloproteinases (MMP). PEA3, an ETS family transcription factor, is elevated in advanced and metastatic ovarian cancer and regulates MMPs in various cell types, therefore, we investigated whether PEA3 is required for the EGFR-dependent induction of MMP mRNA. MMP-9 and MMP-14 mRNA levels were selectively increased in response to EGFR activity in ovarian tumor cells. EGFR activation resulted in nuclear accumulation of PEA3 and direct binding of PEA3, but not the related protein ETS-1, to the endogenous MMP-9 and MMP-14 promoters. Furthermore, PEA3 overexpression was sufficient to induce MMP-9 and MMP-14 mRNA, tumor cell migration, and invasion, suggesting that PEA3 is an important contributor to the metastatic phenotype. Additionally, inhibition of PEA3 expression via short interfering RNA reduced the EGF induction of MMP-9 and MMP-14 gene expression by 92% and 50%, respectively, and impaired EGF-stimulated tumor cell invasion. These results suggest that PEA3 is regulated by EGFR and that the elevated PEA3 expression detected in human ovarian cancer may divert cells to a more invasive phenotype by regulating MMP-9 and MMP-14.

Topics: Cell Line, Tumor; Cell Movement; Cell Nucleus; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 14; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Ovarian Neoplasms; Promoter Regions, Genetic; Protein Binding; RNA, Messenger; Transcription Factors

PLA2 (phospholipase A2) enzymes play critical roles in membrane phospholipid homoeostasis and in generation of lysophospholipid growth factors. In the present study, we show that the activity of the cytosolic iPLA2 (calcium-independent PLA2), but not that of the calcium-dependent cPLA2 (cytosolic PLA2), is required for growth-factor-independent, autonomous replication of ovarian carcinoma cells. Blocking iPLA2 activity with the pharmacological inhibitor BEL (bromoenol lactone) induces cell cycle arrest in S- and G2/M-phases independently of the status of the p53 tumour suppressor. Inhibition of iPLA2 activity also leads to modest increases in apoptosis of ovarian cancer cells. The S- and G2/M-phase accumulation is accompanied by increased levels of the cell cycle regulators cyclins B and E. Interestingly, the S-phase arrest is released by supplementing the growth factors LPA (lysophosphatidic acid) or EGF (epidermal growth factor). However, inhibition of iPLA2 activity with BEL remains effective in repressing growth-factor- or serum-stimulated proliferation of ovarian cancer cells through G2/M-phase arrest. Down-regulation of iPLA2b expression with lentivirus-mediated RNA interference inhibited cell proliferation in culture and tumorigenicity of ovarian cancer cell lines in nude mice. These results indicate an essential role for iPLA2 in cell cycle progression and tumorigenesis of ovarian carcinoma cells.

Topics: Animals; Apoptosis; Blotting, Western; Calcium; Cell Division; Cell Proliferation; Cyclins; Cytosol; Epidermal Growth Factor; Female; G2 Phase; Group VI Phospholipases A2; Humans; Lysophospholipids; Mice; Mice, Inbred BALB C; Mice, Nude; Naphthalenes; Ovarian Neoplasms; Phospholipases A; Phospholipases A2; Pyrones; RNA, Small Interfering; S Phase; Tumor Cells, Cultured

CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been reported to mediate the metastasis of many solid tumors including ovarian, breast, lung and prostate. The over-expression of the epidermal growth factor receptor (EGFR) is associated with the majority of ovarian cancer and has been implicated in the process of malignant transformation by promoting cell proliferation, survival, and motility. In this research, the result first showed that epidermis growth factor (EGF) enhanced the expression of CXCR4 and the migration of ovarian cancer cells, moreover, both stromal cell derived factor-1alpha (SDF-1alpha) and EGF-induced high matrix metallopeptidase 9 (MMP9) expressions. Molecular analysis indicated that augmented CXCR4 and MMP9 expression was regulated by phosphatidylinositol-3-kinase(PI3K)/Akt signal transduction pathway. These results suggested a possible important "cross-talk" between CXCR4 and EGFR intracellular pathways that might link signals of tumor deteriorated and provided a plausible explanation for the poor overall survival rate of patients whose co-expression of CXCR4 and EGFR was detected in their tissue sections. It enlightened that, compared to the respective inhibition of the EGFR or CXCR4 signaling, the simultaneous inhibition of them might be a more useful therapeutic strategy of cancer.

Topics: Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Matrix Metalloproteinase 9; Neoplasm Metastasis; Ovarian Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, CXCR4; Signal Transduction

Ovarian cancer is the primary cause of death from gynecological malignancies with a poor prognosis characterized by widespread peritoneal dissemination. However, mechanisms of invasion and metastasis in ovarian cancer remain poorly understood. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) are often both overexpressed and contribute to the growth of ovarian cancer by activating autocrine pathways. In the present study, we investigated the mechanisms of invasive activity of EGF, HGF, and their synergistic effects in human ovarian cancer cells. Here our data suggest that EGF and HGF may use unique and overlapping signaling cascades leading to the invasive phenotype. We revealed that HGF-mediated cell migration and invasion required the coordinate activation of the phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2. Although EGF-dependent invasive phenotype appeared to have similar requirements for phosphatidylinositol 3-kinase, this growth factor used the alternative p38 MAPK pathway for cell invasion. A significant role of p38 MAPK was further supported by the observation that expression of dominant negative p38 MAPK likewise inhibited EGF-dependent invasiveness and cell motility. We also showed that EGF cooperated with HGF to promote a highly invasive phenotype via the increased secretion of matrix metalloproteinase (MMP)-9. The coincident induction of MMP-9 was functionally significant because inclusion of MMP-9 inhibitor or an anti-MMP-9 neutralizing antibody abolished EGF- and HGF-induced cellular invasion. These findings provide insights into the mechanism of the malignant progression of ovarian cancer.

Topics: Carcinoma; Cell Movement; Collagen; Drug Combinations; Drug Evaluation, Preclinical; Drug Synergism; Epidermal Growth Factor; Female; Hepatocyte Growth Factor; Humans; Laminin; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Ovarian Neoplasms; p38 Mitogen-Activated Protein Kinases; Proteoglycans; Tumor Cells, Cultured

The EGFR is expressed in malignant ovarian tumor tissue, and tissue content of EGFR has been directly associated with poor prognosis in patients with ovarian cancer. The uPA system plays a role in pericellular proteolysis, cell migration, invasion, and is over-expressed in ovarian cancer. This study explored the effects of EGF on uPAR expression in the ovarian cancer cell line OVCAR-3.. We used OVCAR-3 cells and the following methods: cell migration assay, time-lapse video microscopy, real-time PCR, assays for cellular binding of 125I-uPA and cellular degradation of 125I-uPA:PAI-1 complex, biosynthetic labeling using 35S-methionin, Western blot, Northern blot, and ELISAs for uPA, PAI-1, and uPAR.. EGF up-regulates both protein and mRNA not only for uPAR, but also for the ligand uPA and its inhibitor PAI-1. Cell surface uPAR, in control as well as EGF-stimulated cells, is present only in the intact, not the cleaved, form. Ligand binding experiments showed an increase of endogenously occupied uPAR, whereas non-occupied receptor sites were not increased. In addition, EGF treatment resulted in decreased degradation of radiolabeled uPA:PAI-1 complex. This suggests decreased internalization of uPAR, since the complex is internalized together with uPAR. Like EGF, colchicine, which inhibits endocytosis, increased cell surface expression of uPAR. In addition, we found an immediate increase of uPAR after exposing the cells to EGF and this was accompanied by a transient increase of cell migration. The increase of cell surface uPAR in response to EGF is accompanied by increased release of the soluble form of uPAR (suPAR) to the medium as well as by increased cell migration. Both uPAR and suPAR increased in cells treated with the endocytosis inhibitor colchicine even though cell migration was inhibited, suggesting that the mechanism of uPAR shedding is not related to cell migration.. Increased cell surface uPAR in response to EGF stimulation results from mobilization of uPAR from detergent-resistant domains, increased expression of uPAR mRNA, and decreased internalization and degradation of uPAR. Both the anti-uPAR antibody R3, which inhibits binding of uPA, and the EGFR phosphorylation inhibitor Iressa inhibited cell migration in response to uPA as well as to EGF, suggesting that EGFR and uPAR are engaged in the same multiprotein assembly on the cell surface.

Topics: Adenocarcinoma; Blotting, Western; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger

The role of progestogens in the genesis of ovarian cancer remains unclear although a rather protective behaviour has been suggested. Epidemiological studies indicate a possible increase in the risk for combined estrogen/progestin as compared to estrogen alone. It is ambigious whether a difference exists within the various progestogens. Apart from sex steroids, growth factors play a crucial role in the genesis of ovarian cancer, although as yet little investigated. In the present study we have explored the effect of progesterone (P), medroxyprogesterone acetate (MPA) and norethisterone (NET) on the proliferation of human ovarian cancer cells alone and in the presence of growth factors.. For the experiments human ovarian cancer cells (OVCAR-3) were used. The progestogens were tested at the concentrations of 0.01 to 10 microM. The growth factor mixture consisted of EGF, FGF and IGF-I, each at a concentration of 10 pM. The incubation time was three or seven days. Proliferation rate was measured by an ATP-assay.. After three days' incubation the growth factors induced an increase in the proliferation rate of about 50%. Progesterone alone did not show any significant change as compared to the control values, whereas NET and MPA elicited a significant increase at 1 and 10 microM and at 1 microM, respectively. In the presence of growth factors none of the progestogens was able to inhibit the proliferative stimulation. After seven days' incubation the growth factors still showed an increase of about 50%. MPA alone had an inhibitory effect at 10 microM, for NET and P no effects were observed. Again in the presence of growth factors no progestogen was able to show an inhibitory effect.. Our results indicate that progestogens do not have a protective role on the growth of pre-existing ovarian cancer cells, at least in the presence of growth factors. Further investigations are worthwile to evaluate possible differences between the effect of the various progestogens.

Topics: Analysis of Variance; Antineoplastic Agents, Hormonal; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Female; Fibroblast Growth Factors; Humans; In Vitro Techniques; Insulin-Like Growth Factor Binding Proteins; Medroxyprogesterone Acetate; Norethindrone; Ovarian Neoplasms; Progestins; Protective Agents

There is growing evidence that deregulation of E2F transcription factors is causatively involved in the patho-physiology of various tumors. However, no data on the role of E2F family members in tumor biology of ovarian cancer are available. We here describe an expression study of all known E2F transcription factors and their coactivators DP-1 and DP-2 in various human ovarian cancer cell lines and the breast cancer cell line T47D and their involvement in pathways affected by interferon-gamma and EGF. A significant overexpression of the proliferation-promoting E2F1 and especially E2F2 points to a pivotal role in modulating the uncontrolled proliferation in ovarian cancer cells. Of special note is the fact that interferon-gamma treatment did not only caused a reduction of the proliferation-promoting transcription factors E2F1 and E2F2, but also increased the inhibiting transcription factors E2F4 and E2F5, thus underlining the importance of an E2F cross-talk in the anti-proliferative function of interferon-gamma. Moreover, an increase in DP-1 and E2F3 is probably involved in the proliferation-enhancing effect of EGF. Our study provides a new insight in the crucial role of E2F cross-talk, especially the role of the inhibiting transcription factors E2F4 and E2F5, in the tumor biology of cancer and its possible usefulness as targets in anti-cancer therapy.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; E2F Transcription Factors; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; Ovarian Neoplasms; RNA, Messenger; Transcription Factor DP1; Transcription Factors; Transcriptional Activation

Ovarian cancer remains the leading cause of fatality among all gynecologic cancers, although promising therapies are in the making. It has been speculated that metastasis is critical for ovarian cancer, and yet the molecular mechanisms of metastasis in ovarian cancer are poorly understood. Growth factors have been proven to play important roles in cell migration associated with metastasis, and inhibition of growth factor receptors and their distinct cell signaling pathways has been intensively studied, and yet the uncovered interaction or crosstalk among various growth factor receptors complicates this otherwise promising approach. We investigated the crosstalk between EGFR and TrkB, both of which have been known to be important in cell survival and migration in response to EGF and BDNF. Our results showed that both EGF and BDNF induced cell migration and cell proliferation in cultured human ovarian cancer cells (Caov3 cell line). EGF and BDNF transactivated TrkB and EGFR respectively, and activated downstream cell survival components such as Akt. EGFR and TrkB kinase inhibitors inhibited EGF- and BDNF-induced TrkB and EGFR activation and Akt phosphorylation, and cell proliferation and migration. Using EGFR knockout cells, we further demonstrated that EGFR is required for EGF-induced cell migration. Collectively, our data indicate that EGFR and TrkB crosstalk each other in response to EGF and BDNF, leading to cell survival pathway activation in ovarian cancer cells. Our data suggest that a combination of inhibitors of both receptors with cell survival pathway inhibitors would provide a better outcome in the clinical treatment of ovarian cancer.

Topics: Brain-Derived Neurotrophic Factor; Cell Movement; Cell Proliferation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptor, trkB; Transcriptional Activation; Tumor Cells, Cultured

The majority of human endometrial and ovarian cancers express receptors for GnRH type I (GnRH-I). Their proliferation is time- and dose-dependently reduced by GnRH-I and its analogs. GnRH-I analogs activate a phosphotyrosine-phosphatase (PTP) and inhibit EGF-induced mitogenic signal transduction. Recently we found that GnRH type II (GnRH-II) and its agonist [D-Lys6]GnRH-II also have antiproliferative effects on these tumor cells which are significantly greater than those of GnRH-I agonists. In a more recent study, we showed that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. The underlying signal transduction mechanisms of GnRH-II are still unknown. In this study we showed that the mitogenic effects of growth factors in endometrial and ovarian cancer cell lines were counteracted by GnRH-II agonist [D-Lys6]GnRH-II, indicating an interaction with the mitogenic signal transduction. We showed that [D-Lys6]GnRH-II reduces EGF-induced auto-tyrosine-phosphorylation of EGF-receptors via activation of a PTP and that EGF-induced activation of mitogen-activated protein kinase was blocked in cells treated with [D-Lys6]GnRH-II. Furthermore, EGF-induced expression of the immediate early gene c-fos was inhibited by treatment with [D-Lys6]GnRH-II. After knock-out of GnRH-I receptor expression, GnRH-II agonist [D-Lys6]GnRH-II still activated PTP and inhibited the EGF-induced mitogenic signal transduction. These data indicate, that the effects of GnRH-II are not due to a cross-reaction with the GnRH-I receptor. In conclusion these data suggest that the signaling of GnRH-II agonist [D-Lys6]GnRH-II is comparable to that of GnRH-I analogs.

Topics: Endometrial Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Humans; MAP Kinase Kinase Kinases; Ovarian Neoplasms; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-fos; Receptors, LHRH; RNA, Messenger; Signal Transduction; Triptorelin Pamoate

BAG-1 is a multi-functional protein that exists in three major isoforms, BAG-1 p50, p46, and p36. A fourth isoform of 29 kDa also exists but its function remains mostly unknown. To further understand the role of this smaller isoform in ovarian cancer cells, the SKOV3 cell line was transfected with a doxycycline-inducible human BAG-1 p29 isoform or control plasmid. Ovexpression of BAG-1 p29 promotes protection from apoptosis in the presence of EGF as shown by decreased cell death measured by XTT assay and caspase-3 activity. Unexpectedly, however, BAG-1 p29 does not associate with the EGF receptor. When BAG-1 p29 transfectants were incubated in hydrogel-coated plates, BAG-1 p29-expressing SKOV3 cells were significantly more resistant to anoikis as compared to controls, and this correlated with decreased activation of caspase-3. The results of this study implicate BAG-1 p29 in the regulation of both the EGF signaling cascade and the apoptotic cascade induced by loss of anchorage.

Topics: Anoikis; Antineoplastic Agents; Apoptosis; Carrier Proteins; Cell Line, Tumor; Cell Survival; DNA-Binding Proteins; Dose-Response Relationship, Drug; Doxycycline; Drug Interactions; Drug Resistance; Epidermal Growth Factor; Female; HeLa Cells; Humans; Ovarian Neoplasms; Transcription Factors

Epidermal growth factor receptor (EGFR) has been implicated in tumour growth and extension of ovarian cancer. Peritoneal fluid in ovarian cancer patients contains various growth factors that can promote tumour growth and extension. In order to investigate the clinical significance of EGFR ligands as activating factors of ovarian cancer, we examined the cell proliferation-promoting activity and the level of EGFR ligands in peritoneal fluid obtained from 99 patients. Proliferation-promoting activity in peritoneal fluid from 63 ovarian cancer patients (OVCA) was much higher than peritoneal fluid from 18 ovarian cyst patients (OVC) and 18 normal ovary patients (NO), and the activity was suppressed only by antibodies against EGFR or heparin-binding epidermal growth factor (HB-EGF). A large difference was observed in the level of EGFR ligands between HB-EGF and TGF-alpha or amphiregulin. The concentration of HB-EGF in OVCA significantly increased compared to that in OVC or NO (P<0.01). No significant difference in the concentration of TGF-alpha and amphiregulin was found between the OVCA and NO or OVC groups. In peritoneal fluid, HB-EGF is sufficiently elevated to activate cancer cells even at an early stage of OVCA. These results suggested that HB-EGF in peritoneal fluid might play a key role in cell survival and in the proliferation of OVCA.

Topics: Amphiregulin; Ascitic Fluid; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; EGF Family of Proteins; Epidermal Growth Factor; Female; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Middle Aged; Ovarian Neoplasms; Transforming Growth Factor alpha

Topics: ADAM Proteins; ADAM17 Protein; Antineoplastic Agents; Epidermal Growth Factor; Female; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lysophospholipids; Models, Biological; Ovarian Neoplasms; Signal Transduction

Lysophosphatidic acid, which is enriched in the peritoneal fluid of ovarian cancer patients, plays a key role in the progression of ovarian cancer. Lysophosphatidic acid can generate epidermal growth factor receptor (EGFR) signal transactivation involving processing of EGFR ligands by ADAM (a disintegrin and metalloprotease) family metalloproteases. We aimed to investigate the clinical significance of EGFR ligands and ADAM family in the lysophosphatidic acid-induced pathogenesis of ovarian cancer.. We examined the expression of EGFR ligands and ADAM family members in 108 patients with normal ovaries or ovarian cancer, using real-time PCR, immunohistochemistry, and in situ hybridization, and analyzed the clinical roles of these molecules. Statistical analyses of these data were done using the Mann-Whitney test, Kaplan-Meier method, or Spearman's correlation analysis.. Large differences in expression were found for heparin-binding EGF-like growth factor (HB-EGF) and other EGFR ligands and for ADAM 17 and other ADAM family members. HB-EGF expression was significantly increased in advanced ovarian cancer compared with that in normal ovaries (P < 0.01). HB-EGF expression was significantly associated with the clinical outcome (P < 0.01). ADAM 17 expression was significantly enhanced in both early and advanced ovarian cancer compared with that in normal ovaries (both P < 0.01), although it had no clinical significance in the progression-free survival. HB-EGF expression was significantly correlated with ADAM 17 expression (gamma = 0.437, P < 0.01).. Our findings suggest that HB-EGF and ADAM 17 contribute to the progression of ovarian cancer and that HB-EGF plays a pivotal role in the aggressive behavior of a tumor in ovarian cancer.

Topics: ADAM Proteins; ADAM17 Protein; Amphiregulin; Betacellulin; Disease Progression; Disease-Free Survival; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Ovarian Neoplasms; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor alpha

We examined the cAMP-mediated regulation of the epidermal growth factor-like growth factor amphiregulin (AR) in T cells and observed a strong cAMP-induced up-regulation of AR mRNA in a time- and concentration-dependent manner independent of T cell activation. This regulation may be mediated in part through activation of a cAMP-responsive element in the AR promoter, because the cAMP-responsive element conferred cAMP responsiveness to a luciferase reporter in Jurkat TAg cells. Similar effects of AR mRNA induction were seen in T cells treated with cAMP-elevating agents such as prostaglandin E(2) and forskolin as well as with the phosphodiesterase inhibitors rolipram and isobutylmethylxanthine. Furthermore, the induction of AR mRNA by cAMP was strongly suppressed by a protein kinase A type I-selective inhibitor, whereas treatment with an exchange protein directly activated by cAMP-specific agonist did not increase AR levels. In addition, an increase in AR gene transcripts by cAMP was seen in MCF-7 mammary carcinoma cells and H295R adrenal cells. Moreover, the potent cAMP-mediated induction of AR mRNA resulted in increased secretion (5-fold) of AR from T cells. Furthermore, supernatants from cAMP-stimulated T cells containing secreted AR induced phosphorylated MAPK in OVCAR-3 carcinoma cells. In conclusion, our data suggest that AR is under strong regulation by the cAMP pathway in various cell types, and that prostaglandin E(2)- and cAMP-induced AR secretion from T cells may be highly relevant in a microenvironment consisting of tumor cells and infiltrated immune cells, because AR by activating the MAPK pathway through a paracrine route may contribute to proliferation of tumor cells and thus add to neoplastic processes.

Topics: Adenylyl Cyclases; Adrenal Glands; Amphiregulin; Breast Neoplasms; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Dinoprostone; EGF Family of Proteins; Epidermal Growth Factor; Female; Gene Expression; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Male; MAP Kinase Signaling System; Ovarian Neoplasms; Phosphorylation; Promoter Regions, Genetic; Prostatic Neoplasms; RNA, Messenger; T-Lymphocytes; Tumor Cells, Cultured

Gonadotropins play a crucial role in ovarian homeostasis and fertilization through the activation of the cAMP cascade. However, gonadotropin hyper-stimulation may be associated with higher risk for ovarian cancer development. It has been suggested, that high gonadotropin levels in peritoneal and ovarian cystic fluids of patients suffering from benign ovarian cysts, may lead to malignancy. Moreover, we have recently discovered that gonadotropin stimulation can activate the MAPK cascade in target cells. Using DNA microarray technology and RNA from human granulosa cells, we discovered that stimulation with saturating doses of gonadotropins dramatically elevates activity of genes coding for epiregulin and amphiregulin. These gene products can bind and activate the EGF receptor and ERBB4, which are associated with the development of various cancers such as ovarian, breast endometrial and other non-gynecological malignancies. Gonadotropin receptors are expressed not only in the gonads, but also in non-gonadal tissues and in cancer cells. The discovery that gonadotropins activate certain mitogenic signal transduction pathways, may serve as a guide for novel anti-cancer therapy by (1) specific interference at the receptor level to block the gonadotropic response, or arresting the receptor expression and (2) blocking downstream mitogenic signals generated by these hormones, like attenuation of the expression of epiregulin and amphiregulin that belong to the EGF family, using anti-sense and/or SiRNA techniques targeted to suppress their expression. Moreover, since amphiregulin and epiregulin act as mediators of luteinizing hormone (LH) action in the mammalian ovulatory follicles, regulation of the expression of these factors may open new possibilities in treatment of ovarian malfunction implicated with ovarian hyper-stimulation.

Topics: Amphiregulin; Antineoplastic Agents; Cell Transformation, Neoplastic; Drug Design; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; Female; Gene Expression; Glycoproteins; Gonadotropins; Humans; Intercellular Signaling Peptides and Proteins; Luteinizing Hormone; Ovarian Neoplasms; Signal Transduction; Transforming Growth Factor alpha

Ovarian cancer is the most frequent cause of cancer death among all gynecologic cancers. We demonstrate here that lysophosphatidic acid (LPA)-induced ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF) is a critical to tumor formation in ovarian cancer. We found that among the epidermal growth factor receptor (EGFR) family of growth factors, HB-EGF gene expression in cancerous tissues and HB-EGF protein levels in patients' ascites fluid were significantly elevated. The human ovarian cancer cell lines SKOV3 and RMG-1 form tumors in nude mice. Tumor formation of these cells was enhanced by exogenous expression of pro-HB-EGF and completely blocked by pro-HB-EGF gene RNA interference or by CRM197, a specific HB-EGF inhibitor. Transfection with mutant forms of HB-EGF indicated that the release of soluble HB-EGF is essential for tumor formation. LPA, which is constitutively produced by ovarian cancer cells, induced HB-EGF ectodomain shedding in SKOV3 and RMG-1 cells, resulting in the transactivation of EGFR and the downstream kinase extracellular signal-regulated kinase/mitogen-activated protein kinase. LPA-induced transactivation was abrogated by HB-EGF gene RNA interference or by CRM197. Introduction of lipid phosphate phosphohydrolase, which hydrolyzes LPA, decreased the constitutive shedding of HB-EGF, EGFR transactivation, and the tumorigenic potential of SKOV3 and RMG-1 cells. These results indicate that HB-EGF is the primary member of the EGFR family of growth factors expressed in ovarian cancer and that LPA-induced ectodomain shedding of this growth factor is a critical step in tumor formation, making HB-EGF a novel therapeutic target for ovarian cancer.

Topics: Animals; Bacterial Proteins; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Extracellular Fluid; Female; Gene Expression; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lysophospholipids; Mice; Ovarian Neoplasms; Receptors, Cell Surface; Transcriptional Activation; Transfection

Development of targeted therapeutics for ovarian cancer requires a basic understanding of ovarian epithelial carcinoma cell biology. The role of estrogen and epidermal growth factor (EGF) in control of cell growth was investigated in a panel of ovarian carcinoma lines.. EGF receptor (EGFR) was detected by flow cytometry and estrogen receptor (ER) by Northern blot. Western blotting and [(3)H]thymidine incorporation were used to determine receptor activation and the effects of ligand exposure and growth factor antagonists, including the antineoplastic agent, suramin, on cell growth.. Only one cell line, OV266, expressed ER and responded to beta-estradiol with increases in DNA synthesis and cell proliferation that could be blocked by the pure antiestrogen ICI 182,780. All cell lines possessed functional EGFR as measured by flow cytometry and phosphorylation of the receptor and mitogen-activated protein kinase after EGF treatment, but only two cell lines (OV177 and OV266) proliferated in response to exogenous EGF. Suramin had limited effectiveness in inhibiting growth in four of five cell lines and had a striking dose-dependent stimulatory effect on OV266 cell growth. The proliferative response to suramin could be inhibited with EGFR antagonists.. Cultured epithelial ovarian carcinoma cell lines express EGFR (5/5) and can express ER (1/5). Differential growth responses to EGF were observed despite uniform receptor and MAPK activation. Unexpectedly, the antineoplastic agent suramin increased growth of ER positive ovarian carcinoma cells in an EGFR-dependent manner. These studies provide insight into the complex interactions of these systems in control of ovarian cancer cell growth.

Topics: Antineoplastic Agents; Cell Division; Cell Line, Tumor; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Estradiol; Female; Flow Cytometry; Humans; Ovarian Neoplasms; Progesterone; Receptors, Estrogen; Signal Transduction; Somatomedins; Stimulation, Chemical; Suramin; Testosterone

It has been previously demonstrated that human ovarian cancer cells express FSH receptor (FSHR). However, whether FSHR plays a role in ovarian cancer development is still ambiguous. To investigate the role of FSHR in tumor progression, we overexpressed the receptor in SV40 Tag immortalized ovarian surface epithelium (OSE) cell lines (IOSE-80PC, a postcrisis line, and IOSE-398), which are preneoplastic and nontumorigenic. We compared the expression levels of several selected oncogenes in nontransfected (80PC), vector-transfected (80PCV), FSHR-transfected IOSE (80PCF) cells, and established ovarian cancer cell lines (OVCAR-3 and SKOV-3). Significantly increased protein levels of epithelial growth factor receptor, HER-2/neu, and c-Myc, but not K-Ras, were observed in FSHR-overexpressing 80PCF cells when compared with 80PCV cells. Constitutive phosphorylation of ERK1/2 was augmented in 80PCF cells, whereas phosphorylation of the other MAPK including p38 and Jun N-terminal kinase was unchanged. Considerable constitutive phosphorylation of ERK1/2 was also observed in OVCAR-3 and SKOV-3 cell lines when compared with 80PCV. More importantly, 80PCF cells grew more rapidly than 80PC and 80PCV cells. In conclusion, we have demonstrated that FSHR was highly expressed in OVCAR-3 and 80PCF cells transfected with FSHR overexpression vector. The 80PCF cell line showed increased levels of epithelial growth factor receptor, HER-2/neu, and c-myc and constitutive activation of ERK1/2. The rate of proliferation of the 80PCF cells was increased, compared with control cell lines. These results suggest that the overexpression of FSHR may be associated with enhanced levels of potential oncogenic pathways and increased proliferation in preneoplastic ovarian surface epithelial cells.

Topics: Cell Division; Cells, Cultured; Epidermal Growth Factor; Epithelial Cells; Female; Follicle Stimulating Hormone; Humans; Mitogen-Activated Protein Kinases; Oncogenes; Ovarian Neoplasms; Precancerous Conditions; Receptors, FSH; Transfection

The aim of this study was to determine the effects of exogenous expression of the catalytic subunit of telomerase (hTERT) on the lifespan, growth characteristics, and tumorigenicity of normal human ovarian surface epithelial (OSE) cells.. Low-passage primary cultures of normal human OSE cells were transfected with hTERT and the resulting cell lines were characterized.. The ectopic expression of hTERT stabilized the telomeres of the OSE cultures above 8 kb. The hTERT-transfected OSE cell lines grew beyond the normal lifespan seen in OSE cells and propagated in culture for more than 40 passages before senescing. Moreover, the hTERT-transfected cells demonstrated extensive proliferative capacity as evidenced by their ability to continuously grow even when seeded at low dilutions. The morphologic features and normal differentiation patterns seen in normal OSE cells were likewise retained by the hTERT-transfected cells. In addition, the cultures remained responsive to physiologic concentrations of epidermal growth factor and transforming growth factor-beta. Changes associated with neoplastic transformation like anchorage-independent growth, tumorigencity and karyotypic instability were not observed.. We were able to show that the ectopic expression of hTERT in normal human OSE: 1) resulted in cultures with greater growth potential and longer lifespan and 2) did not induce a transformed phenotype previously seen in viral oncogene-transfected OSE cells. The established cell lines would not only provide sufficient material for comprehensive studies to investigate the normal physiology of OSE cells, but could also help in the understanding of the early steps of ovarian carcinogenesis.

Topics: Animals; Cell Differentiation; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Cells, Cultured; DNA-Binding Proteins; Epidermal Growth Factor; Epithelial Cells; Female; Humans; Immunohistochemistry; Keratins; Mice; Mice, SCID; Ovarian Neoplasms; Ovary; Telomerase; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Emerging data suggest that signaling by heparin-binding growth factors is influenced by the sulfation state of N-acetylglucosamine residues of heparan sulfate proteoglycans (HSPGs). Here we report that the recently identified protein HSulf-1, a heparin-degrading endosulfatase, encodes a cell surface-associated enzyme that diminishes sulfation of cell surface HSPGs. The message encoding this enzyme is readily detectable in a variety of normal tissues, including normal ovarian surface epithelial cells, but is undetectable in 5 of 7 ovarian carcinoma cell lines and markedly diminished or undetectable in approximately 75% of ovarian cancers. Similar down-regulation is also observed in breast, pancreatic, renal cells, and hepatocellular carcinoma lines. Re-expression of HSulf-1 in ovarian cancer cell lines resulted in diminished HSPG sulfation, diminished phosphorylation of receptor tyrosine kinases that require sulfated HSPGs as co-receptors for their cognate ligands, and diminished downstream signaling through the extracellular signal-regulated kinase pathway after treatment with fibroblast growth factor-2 or heparin-binding epidermal growth factor. Consistent with these changes, HSulf-1 re-expression resulted in reduced proliferation as well as sensitivity to induction of apoptosis by the broad spectrum kinase inhibitor staurosporine and the chemotherapeutic agent cisplatin. Collectively, these observations provide evidence that HSulf-1 modulates signaling by heparin-binding growth factors, and HSulf-1 down-regulation represents a novel mechanism by which cancer cells can enhance growth factor signaling.

Topics: 5' Untranslated Regions; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Division; Cloning, Molecular; Epidermal Growth Factor; Epithelial Cells; Female; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Ovarian Neoplasms; Ovary; Recombinant Proteins; Signal Transduction; Sulfotransferases; Transfection; Tumor Cells, Cultured

Alteration in epidermal growth factor receptor (EGFR) family signaling is among the most frequently implicated effectors of human oncogenesis. Overexpression of members of this family of receptors has often been detected in many epithelial tumors and is believed to be associated with an overall poor prognosis in patients with cancer. Therefore, we hypothesized that identification of potential EGF target genes in normal cells will provide a basis for unbiased genetic analysis of this signaling pathway in cancer. We utilized Atlas Rat 1.2 nylon cDNA arrays (Clontech) to determine gene expression changes in normal rat ovarian surface epithelial (ROSE) cells following EGF treatment. The results indicate activation of genes involved in a wide variety of cellular mechanisms, including regulation of cell cycle and proliferation, apoptosis, and protein turnover. In addition, using an in vitro model of ovarian cancer, we demonstrated that malignant transformation of ROSE cells resulted in alteration of downstream effectors of the EGFR pathway, as exemplified by aberrant expression of p66Shc, c-Jun, c-Myc, c-Fos, Lot1, p21Cip/Waf, and cdc25A. These data suggest that knowledge of the downstream genetic lesions, which may result in loss of growth factor requirement of the affected cells, will be crucial for the selection of the EGFR pathway as an effective target for cancer therapy.

Topics: Animals; Cell Size; Cells, Cultured; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Gene Expression Profiling; Gene Expression Regulation; Humans; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Ovary; Rats; Signal Transduction; Tumor Cells, Cultured

The majority of ovarian cancers (OCs) arise from the ovarian surface epithelium (OSE). Proliferation of the OSE can be regulated by a number of autocrine and paracrine factors, including transforming growth factor beta (TGFbeta). Defects in the TGFbeta signaling pathway have been implicated in a number of cancers, including ovarian. We previously found that the TGFbeta signaling pathway is intact and functional in primary human OC cells, and that these cells stop growing in response to TGFbeta. Ovarian cancer cells in vivo are exposed to TGFbeta, yet continue to proliferate, therefore, mechanisms must exist to inhibit TGFbeta signaling contributing to uncontrolled cellular proliferation. Numerous signaling pathways converge with the TGFbeta pathway to modulate its effects, including signaling induced by epidermal growth factor (EGF). We hypothesized that EGF can modulate TGFbeta signaling and contribute to uncontrolled cellular proliferation of OC cells. Our results show that EGF abrogates the antiproliferative effect of TGFbeta. EGF does not modulate TGFbeta signaling by inhibiting receptor-activated Smad (R-Smad) phosphorylation or nuclear translocation. Rather, EGF decreases TGFbeta-induced mRNA expression of the cell cycle regulator, p15(INK4B), contributing to decreased sensitivity of OC cells to the antiproliferative effect of TGFbeta.

Topics: Active Transport, Cell Nucleus; Blotting, Northern; Blotting, Western; Cell Cycle Proteins; Cell Division; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; DNA-Binding Proteins; Epidermal Growth Factor; Female; Humans; Microscopy, Fluorescence; Ovarian Neoplasms; Phosphorylation; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad3 Protein; Time Factors; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins; Up-Regulation

The epidermal growth factor (EGF) receptor is frequently overexpressed in malignant tumors, and its level is correlated with increased cellular resistance to ionizing radiation. However, no precedent studies have investigated whether expression of EGF receptor would by itself confer on cancer cells resistance to radiation. The current study is aimed to address this question.. A full-length human EGF receptor expression vector was transfected into the OCA-I murine ovarian carcinoma cells for stable clones expressing various levels of EGF receptors. Apoptosis and cell clonogenic survival assays were used to evaluate the sensitivity of the resulting cell clones to ionizing radiation.. OCA-I cell clones expressing various levels of EGF receptor (OCA-I EGFR) were obtained. These clones showed an EGF receptor level-dependent increase in resistance to ionizing radiation, measured by apoptosis and cell clonogenic survival assays. Compared with the results for parental OCA-I and control vector-transfected OCA-I cells at the 10% cell survival level, the radioresistance was increased by a factor of 1.60 for EGFR-C5 (high level of EGF receptor expression), 1.37 for EGFR-C3 (intermediate level of EGF receptor expression), and 1.28 for EGFR-C1 (low level of EGF receptor expression). Treatment of the OCA-I EGF receptor transfectants with the anti-EGF receptor monoclonal antibody C225 downregulated the levels of EGF receptor, reduced the phosphorylation levels of EGF receptor downstream substrates (such as Akt and MAPK), and reversed the cellular radioresistance.. Our results demonstrate that overexpression of the EGF receptor conferred cellular resistance to ionizing radiation. The EGF receptor is thus a valid target for potential radiosensitization.

Topics: Animals; Apoptosis; Cloning, Molecular; Dose-Response Relationship, Radiation; Epidermal Growth Factor; Female; Gene Expression Regulation; Humans; Mice; Ovarian Neoplasms; Radiation Dosage; Radiation Tolerance; Statistics as Topic; Transfection; Tumor Cells, Cultured

Cyclooxygenase-2 (COX-2) seems to be involved at various steps in the processes of malignant transformation and tumor progression. Investigations have shown that COX-2 overexpression is associated with increased proliferation, reduced apoptosis, and angiogenesis.. Specimens from 125 patients with high-grade, advanced-stage (Stage III-IV) serous ovarian carcinoma were evaluated by immunohistochemistry for COX-2, p53, bcl-2, epidermal growth factor receptor (EGFR), and Her-2/neu expression and for CD34-stained microvessel density (MVD). Statistical analysis was performed to investigate the correlations between COX-2 expression and 1) clinicopathologic characteristics, 2) tumor MVD, and 3) expression of other molecular markers. The effect of COX-2 expression on survival was determined using survival analysis.. Increased COX-2 expression was significantly correlated with tumor MVD (Spearman rank correlation test: r = 0.41; P < 0.001). There was no association observed between COX-2 expression and expression levels of EGFR, Her-2/neu, bcl-2, or p53. Patients who had tumors that showed high COX-2 expression had a worse prognosis compared with patients who had tumors with low expression (death hazard ratio, 2.0; 95% confidence interval, 1.2-3.5; P < 0.001). A multivariate analysis revealed that COX-2 expression was the strongest predictor of survival among the different prognostic factors analyzed.. The current study demonstrated that COX-2 expression was correlated significantly with survival in patients with high-grade, high-stage serous ovarian carcinoma. Expression of COX-2 also was correlated with tumor angiogenesis but not with EGFR, Her-2/neu, or p53 expression. In addition to their prognostic significance, a better understanding of the biology of these molecular changes may help identify new targets for therapy in patients with ovarian carcinoma.

Topics: Adult; Aged; Biomarkers, Tumor; Biopsy, Needle; Cohort Studies; Cystadenoma, Serous; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Middle Aged; Multivariate Analysis; Neoplasm Staging; Neovascularization, Pathologic; Ovarian Neoplasms; Probability; Prognosis; Prostaglandin-Endoperoxide Synthases; Regression Analysis; Sensitivity and Specificity; Survival Analysis

The abnormal activation of the epidermal growth factor (EGF) pathway is one of the most common findings in human cancer, and a number of molecular devices of laboratory and clinical relevance have been designed to block this transduction pathway. Because of the large number of cellular events that might be regulated through the activation of the four EGF receptor family members, it is possible that screening methodologies for the identification of new molecular targets working downstream of these pathways may provide new tools for cancer diagnosis and potentially prevention and therapy. In searching for EGF target genes, we have identified ERG1.2, the mouse homolog of the solid tumor-associated gene STAG1. Both in humans and in mice, it belongs to a new gene family that can give origin to several protein isoforms through alternative splicing and/or multiple translation starts. Sequence analysis and experimental data suggest that ERG1.2 is likely to function as a membrane-bound protein interacting with downstream signaling molecules through WW- and SH3-binding domains. ERG1.2 is a cell-cycle-regulated gene, and both ERG1.2 and STAG1 are induced by EGF and other growth factors at the transcript and protein levels. Finally, we have demonstrated that, besides prostate cancer and renal cell carcinoma, STAG1 was also overexpressed in breast and ovarian cancer cell lines and in breast primary tumors. Although in most cases STAG1 overexpression is probably due to the abnormal activation of the EGF pathway, we have also demonstrated genetic amplification and rearrangement of its locus in one breast cancer cell line and one primary ovarian cancer, suggesting that STAG1 might be a direct molecular target in the carcinogenetic process. Thus its overexpression might be regarded not only as a tumor marker but also as a potentially pathogenetic event.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Breast Neoplasms; Cell Cycle; Cloning, Molecular; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Gene Amplification; Gene Expression Regulation, Neoplastic; Gene Rearrangement; Growth Substances; Humans; Membrane Proteins; Molecular Sequence Data; Nuclear Proteins; Ovarian Neoplasms; Polymerase Chain Reaction; RNA, Neoplasm; Sequence Homology, Amino Acid; Up-Regulation

The expression of transforming growth factor alpha (TGFalpha), amphiregulin (AR) and cripto-1 (CR-1) was assessed by immunohistochemistry in 83 specimens (59 primary ovarian tumors and 24 extra-ovarian carcinomas) that were obtained from 68 ovarian carcinoma patients. Within the 59 primary tumors, 54 (92%) expressed immunoreactive TGFalpha, 45 (76%) expressed AR, and 28 (47%) expressed CR-1. The expression of AR and CR-1 mRNAs in the ovarian carcinomas was also demonstrated by RT-PCR analysis. Seventeen extra-ovarian specimens (71%) were found to express CR-1, whereas AR and TGFalpha were expressed respectively in 21 (87%) and 22 (92%) extra-ovarian tissues. In 15 cases for whom both ovarian and extra-ovarian tissues were available, a statistically significant higher expression of CR-1 was found in extra-ovarian specimens. A statistically significant correlation was found between AR expression in the ovarian carcinomas and both low grade and low proliferative activity. Finally, expression of TGFalpha was predictive of longer progression-free survival. These data strongly suggest that the EGF-related peptides might be involved in the pathogenesis and outcome of human ovarian cancer.

Topics: Adult; Aged; Amphiregulin; EGF Family of Proteins; Epidermal Growth Factor; Female; Glycoproteins; GPI-Linked Proteins; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Ovarian Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor alpha

Immunohistochemistry was used to determine the expression of granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) in primary ovarian carcinomas. The expression of G-CSFR was observed in the malignant cells of each of the 46 primary carcinomas examined; G-CSF was coexpressed in both the malignant epithelial cells and the stroma of 56.5% of the specimens. Thus the majority of ovarian carcinomas harbor both potential autocrine and paracrine G-CSF axes. In 37% of the samples, G-CSF was expressed only within stromal cells, suggesting that only a potential paracrine system is in place. In a preliminary, retrospective, evaluation, the survival of patients whose tumors expressed only the apparent paracrine loop was significantly worse than patients whose tumors expressed both potential autocrine and paracrine G-CSF-based regulatory loops (14.5 vs. 42.5 months, respectively). Studies on the potential function of G-CSF were performed using the G-CSFR-expressing OVCAR-3 ovarian carcinoma line. As a single agent, rhG-CSF failed to stimulate [3H]-thymidine incorporation in these cells, but enhanced the mitogenic action of epidermal growth factor (EGF) in a dose-dependent manner. Thus, potential autocrine and/or paracrine loops involving G-CSF and its receptor occur in over 90% of primary ovarian carcinomas, and may act to modulate the action of growth factors.

Topics: Cell Survival; Epidermal Growth Factor; Female; Granulocyte Colony-Stimulating Factor; Humans; Immunohistochemistry; Ovarian Neoplasms; Receptors, Granulocyte Colony-Stimulating Factor; Recombinant Proteins; Thymidine; Tumor Cells, Cultured

Activation of the epidermal growth factor (EGF) receptor regulates many processes associated with metastasis, including modulation of cell:cell and cell:substrate interactions, production of matrix-degrading proteinases, and cellular migration. We have demonstrated previously that EGF stimulates migration and matrix metalloproteinase (MMP)-9-dependent invasion of ovarian cancer cells. In this study, we compare the roles of EGF-induced phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) activities in regulation of cellular responses associated with ovarian tumor cell metastasis. Inhibition of PI3K and MAPK activity impairs EGF-stimulated cell migration, in vitro invasion, and MMP-9 production. PI3K activity is not required for growth factor disruption of cell:cell junctions, whereas inhibitors of extracellular signal-regulated kinase (ERK)1/ERK2 activation and p38 MAPK activity block EGF-dependent junction dissolution. EGF promotes pro-MMP-9 binding to the cell surface through a mechanism that is independent of extracellular enzyme concentration. Interestingly, inhibition of PI3K activity abolishes EGF-induced cell surface association of pro-MMP-9, whereas inhibitors of MAPKs only partially block the response. These data suggest that EGF receptor activation promotes a PI3K-dependent induction of a cell surface pro-MMP-9 binding component that may facilitate gelatinase-mediated cellular invasion and supports an expanded role for elevated PI3K activity in cellular responses associated with ovarian tumor metastasis. In addition, our findings support the hypothesis that divergent kinase activities regulate distinct cellular events associated with growth factor-induced invasion of ovarian cancer cells.

Topics: Adherens Junctions; Cell Membrane; Cell Movement; Desmosomes; Epidermal Growth Factor; ErbB Receptors; Female; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Tissue Inhibitor of Metalloproteinase-1; Tumor Cells, Cultured

The signaling pathway through which LHRH acts in endometrial and ovarian cancers is distinct from that in the anterior pituitary. The LHRH receptor interacts with the mitogenic signal transduction of growth factor receptors, resulting in down-regulation of expression of c-fos and proliferation. Only limited data are available on the cross-talk between LHRH receptor signaling and inhibition of mitogenic signal transduction. The present experiments were performed to analyze in endometrial and ovarian cancer cells: 1) whether mutations or splice variants of the LHRH receptor are responsible for differences in LHRH signaling, 2) the coupling of G protein subtypes to LHRH receptor, 3) the phosphotyrosine phosphatase (PTP) activation counteracting growth factor receptor tyrosine kinase activity. For these studies, the well characterized human Ishikawa and Hec-1A endometrial cancer cell lines and human EFO-21 and EFO-27 ovarian cancer cell lines were used, which express LHRH and its receptor. 1) Sequencing of the complementary DNA of the LHRH receptor from position 31 to position 1204, covering the complete coding region (position 56 to position 1042) showed that there are neither mutations nor splice variants of the LHRH receptor transcript in Ishikawa and Hec-1A endometrial cancer cells or in EFO-21 and EFO-27 ovarian cancer cells. 2) All analyzed cell lines except for the ovarian cancer cell line EFO-27 expressed both G proteins, alpha(i) and alpha(q), as shown by RT-PCR and Western blotting. In the EFO-27 cell line only G protein alpha(i), not G protein alpha(q), expression was found. Cross-linking experiments using disuccinimidyl suberate revealed that in the cell lines expressing G protein alpha(i) and G protein alpha(q), both G proteins coupled to the LHRH receptor. Inhibition of epidermal growth factor (EGF)-induced c-fos expression by LHRH, however, was mediated through pertussis toxin (PTX)-sensitive G protein alpha(i). Moreover, LHRH substantially antagonized the PTX-catalyzed ADP-ribosylation of G protein alpha(i). 3) Using a phosphotyrosine phosphatase assay based on molybdate-malachite green, treatment of quiescent EFO-21 and EFO-27 ovarian cancer cells and quiescent Ishikawa and Hec-1A endometrial cancer cells with 100 nM of the LHRH agonist triptorelin resulted in a 4-fold increase in PTP activity (P < 0.001). This effect was completely blocked by simultaneous treatment with PTX, supporting the concept of mediation through G protein alpha(i). As

Topics: Adenosine Diphosphate Ribose; Alternative Splicing; Base Sequence; Cell Division; Endometrial Neoplasms; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Genes, fos; Gonadotropin-Releasing Hormone; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Proteins; Humans; Molecular Sequence Data; Mutation; Ovarian Neoplasms; Pertussis Toxin; Phosphorylation; Phosphotyrosine; Protein Tyrosine Phosphatases; Receptors, LHRH; RNA, Messenger; Sequence Analysis, DNA; Signal Transduction; Tumor Cells, Cultured; Virulence Factors, Bordetella

Activation of the epidermal growth factor (EGF) receptor has previously been shown to increase the sensitivity of cancer cells to DNA-damaging agents, including cisplatin, UV-B, and gamma-radiation. We now investigated the mechanisms by which EGF enhances the sensitivity of human ovarian cancer cells to cisplatin.. The effect of EGF on cisplatin sensitivity could not be entirely explained by alterations in the cellular detoxification of cisplatin by glutathione or DNA repair of transcribed genes, as assessed by a plasmid reactivation assay. Furthermore, EGF did not affect the levels of several proteins that regulate apoptotic pathways, including bcl2, bclXL, bax and p53. Cisplatin treatment resulted in activation of caspase 3 and subsequent cleavage of specific substrates containing the DEVD (Asp-Glu-Val-Asp) amino acid sequence, including PARP (poly(ADP-ribose) polymerase). The EGF-mediated increase in cisplatin-induced apoptosis, however, did not result in increased caspase 3 activity. Moreover, apoptosis induced by cisplatin alone was completely inhibited by the caspase 3 inhibitor DEVD-CHO, whereas cell death induced by combined treatment with cisplatin and EGF was not prevented by inhibition of caspase 3.. Our results suggest that, although cisplatin alone induces apoptosis by a caspase 3 dependent pathway, EGF enhances cisplatin-induced cell death by activating an apoptotic pathway that is independent of caspase 3.

Topics: Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cisplatin; Drug Synergism; Epidermal Growth Factor; Female; Glutathione; Humans; Immunoblotting; Ovarian Neoplasms; Tumor Cells, Cultured

Ezrin is a member of the ezrin, radixin, and moesin family. These proteins are membrane-actin cross-linking proteins. Furthermore, ezrin is an important signal transduction protein that undergoes phosphorylation and translocation on stimulation by growth factors. Ezrin phosphorylation and translocation are thought to be correlated with cell motility, invasion, and carcinoma metastasis. Recently, the authors reported that an ezrin antisense phosphorothionate could significantly inhibit endometrial carcinoma cells' penetration in the Matrigel membrane cancer invasion assay. In the current study, the authors measured ezrin content in clinical ovarian epithelial carcinoma (OVCA) specimens and cell lines and investigated whether interleukin (IL)-1alpha and epidermal growth factor (EGF) induce an invasive phenotype in OVCA cells via ezrin phosphorylation and translocation.. Twenty-five normal ovary, 25 primary OVCA, 21 metastatic OVCA tissue (7 in omentum, 16 in ascites), and 3 OVCA cell lines were collected for Western blot detection of ezrin content. The OVCA cell line SKOV3 was treated with IL-1alpha or EGF. Indirect immunofluorescence staining followed by confocal laser scanning and double-staining electron microscopic immunohistochemistry were used to investigate changes in the intracellular distribution of ezrin and cell morphology after IL-1alpha or EGF treatment. The content of ezrin was measured by Western blotting and analyzed by the National Institutes of Health Image computer program. Immunoprecipitation and Western blot techniques were used for ezrin phosphorylation studies. Genistein was used to block tyrosine phosphorylation.. (1) Ezrin was overexpressed in OVCA, with the highest values in metastases. (2) Interleukin-1alpha and EGF significantly increased OVCA tyrosine phosphorylation, ezrin translocation, and cell growth. (3) These effects were abolished by treatment with the tyrosine kinase inhibitor, genistein. (4) Treatment with IL-1alpha or EGF induced an invasive phenotype, i.e., membrane ruffling, and process formation.. High expression and activation of ezrin appear to be related to OVCA metastatic behavior. Interleukin-1alpha and EGF may regulate OVCA invasive behavior by activating ezrin tyrosine phosphorylation, translocation, and cancer cell proliferation. The authors' results may partially explain why OVCA patients with positive macrophage colony stimulating factor (a chemoattractant of IL-1alpha secreting monocytes) or EGF receptors (c-erb B-2) have a poor prognosis.

Topics: Blotting, Western; Carcinoma; Cell Division; Cytoskeletal Proteins; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-1; Neoplasm Invasiveness; Neoplasm Metastasis; Ovarian Neoplasms; Phenotype; Phosphoproteins; Phosphorylation; Translocation, Genetic; Tumor Cells, Cultured; Tyrosine

Over-expression of the ErbB-2 proto-oncogene frequently coincides with an aggressive clinical course of certain human adenocarcinomas. The ErbB-2 receptor is a member of the ErbB family of growth factor receptors, and within this complex signaling network, ErbB-2-containing heterodimers are preferentially formed. To assess whether ErbB-2 is a critical component in epidermal growth factor (EGF)-mediated stimulation of tumor cell proliferation, we used as a model SK-OV-3 ovarian cancer cells, which over-express EGF receptor (EGFR) and ErbB-2 receptors. In these cells, we reduced ErbB-2 mRNA and protein expression by transfection with ErbB-2-targeted hammerhead ribozymes and generated cell lines expressing different levels of ErbB-2. In SK-OV-3 cells, ErbB-2 expression conferred a growth advantage and soft agar experiments revealed that ErbB-2 was rate-limiting for anchorage-independent growth. The induction of colony formation by EGF was completely abrogated in ErbB-2-depleted cells, despite unchanged expression levels and tyrosine phosphorylation of the EGFR. The duration of EGF-mediated c-Fos mRNA up-regulation was decreased in parallel with loss of ErbB-2 expression. Furthermore, the rate of spontaneous apoptosis was increased in ErbB-2-depleted cells. Our results demonstrate that in human ovarian cancer cells the EGFR-ErbB-2 heterodimer, and not the EGFR homodimer, can be rate-limiting for EGF-mediated proliferation, thus suggesting that the oncogenic activity of ErbB-2 in human tumors is due in part to its ability to increase the growth response to stroma-derived EGF-like growth factors.

Topics: Apoptosis; Cell Division; Epidermal Growth Factor; ErbB Receptors; Female; Glycoproteins; Humans; Neuregulin-1; Ovarian Neoplasms; Proto-Oncogene Mas; Proto-Oncogene Proteins c-fos; Receptor, ErbB-2; RNA, Catalytic; Tumor Cells, Cultured

The epidermal growth factor (EGF)-like peptides CRIPTO (CR), amphiregulin (AR) and transforming growth factor alpha (TGFalpha) are expressed in human ovarian carcinomas.. The expression of AR, CR and TGFalpha in ovarian carcinoma cell lines was assessed by immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). The antiproliferative effects of antisense phosphorothioate oligodeoxynucleotides (AS S-Oligos) directed against either AR, CR or TGFalpha was evaluated by using a clonogenic assay.. A majority of the ovarian carcinoma cell lines was found to express TGFalpha, AR and CR mRNAs and proteins. AS S-Oligos directed against either AR, CR or TGFalpha were able to inhibit the anchorage-independent growth of NIH:OVCAR3 and NIH:OVCAR8 cells in a dose dependent manner. A 30%-50% growth inhibition was observed at a 2 microM concentration of the AS S-Oligos. Treatment of these cells with combinations of EGF-related AS S-Oligos resulted in a more significant growth inhibition when compared to treatment with a single AS S-oligo. A 60%-75% growth inhibition was observed using combinations of AR, CR and TGFalpha AS S-oligos at a total concentration of 2 microM. An additive growth-inhibitory effect occurred when ovarian carcinoma cells were exposed to the AS S-Oligos after treatment with either paclitaxel or cis-platinum.. These data suggest that EGF-related peptides function as autocrine growth factors in ovarian carcinoma cells, and that they might represent targets for experimental therapy of ovarian carcinoma.

Topics: Amphiregulin; Carcinoma; EGF Family of Proteins; Epidermal Growth Factor; Female; Glycoproteins; Growth Substances; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Oligonucleotides, Antisense; Ovarian Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thionucleotides; Transforming Growth Factor beta; Tumor Cells, Cultured

Greater than 95% of ovarian cancers originate from the epithelial cells on the surface of the ovary termed ovarian surface epithelium (OSE). A normal aspect of OSE function is repeated proliferation after ovulation, and this is postulated to be involved in part in the onset of ovarian cancer. The hypothesis tested is that locally produced growth factors have an important role in controlling OSE proliferation. The current study investigates the potential role of the growth factor kit ligand (KL)/stem cell growth factor and its receptor c-kit in normal OSE biology and ovarian cancer. Human tumors from borderline, stage I, and stage III cases of ovarian cancer were found to express KL and c-kit protein in the epithelial cell component by ICC analysis. The stromal cell component of human ovarian tumors contained little immunostaining. Bovine ovarian physiology and endocrinology are similar to the human such that cow ovaries were used as a model system to investigate normal OSE functions. KL and c-kit proteins were detected in the OSE from both normal human and bovine ovaries. Adjacent ovarian stromal tissue contained less intense but positive KL and c-kit immunostaining. To extend the ICC results, RNA was collected from normal bovine OSE and ovarian stromal cells to examine KL gene expression. KL transcripts were detected in cultured OSE and stromal cells by Northern blot analysis. KL gene expression was found to be high in freshly isolated OSE but low in freshly isolated stroma using a quantitative polymerase chain reaction procedure. Levels of KL gene expression in cultured OSE and stroma increased to high levels. Observations indicate that normal OSE expresses high levels of KL in vivo and in vitro. The actions of KL on the growth of both normal OSE cells and ovarian cancer cells was investigated. KL was found to stimulate the growth of normal OSE cells in a similar manner to epidermal growth factor. Observations demonstrate the production and action of KL by normal OSE cells and ovarian cancer cells. Coexpression of KL and c-kit by normal OSE suggests that KL can act as an autocrine factor for OSE. The local production and action of KL on OSE provides insight into normal OSE biology, and a factor that may be involved in the onset and progression of ovarian cancer.

Topics: Animals; Cattle; Cell Division; Epidermal Growth Factor; Epithelial Cells; Female; Gene Expression; Humans; Immunohistochemistry; Ovarian Neoplasms; Ovary; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stem Cell Factor

Ascitic fluid and plasma from ovarian cancer patients, but not from patients with nongynecological tumors, contain elevated levels of the bioactive phospholipid lysophosphatidic acid (LPA). We show that ovarian cancer cells constitutively produce increased amounts of LPA as compared with normal ovarian epithelium, the precursor of ovarian epithelial cancer, or breast cancer cells. In addition, LPA, but not other growth factors, increases LPA production by the OVCAR-3 ovarian cancer cell line but not by normal ovarian epithelium or breast cancer cell lines. We show that phospholipase D activity contributes to both constitutive and LPA-induced LPA production by ovarian cancer cells. Constitutive and LPA-induced LPA synthesis by ovarian cancer cells is differentially regulated with respect to the requirement of specific phospholipase A2 (PLA2) subgroups. Group IB (pancreatic) secretory PLA2 plays a critical role in both constitutive and LPA-induced LPA formation, whereas group IIA (synovial) secretory PLA2 contributes to LPA-induced LPA production only. Calcium-dependent and/or -independent cytosolic PLA2s are required for constitutive LPA synthesis but do not play a role in LPA-induced LPA formation. LPA increases the proliferation of ovarian cancer cells, decreases sensitivity to cisplatin, the most commonly used drug in ovarian cancer, decreases apoptosis and anoikis, increases protease production, and increases production of neovascularization mediators. Thus, an understanding of the source and regulation of LPA production in ovarian cancer patients could identify novel targets for therapy.

Topics: Antineoplastic Agents; Blotting, Northern; Breast Neoplasms; Calcium; Cell Division; Cell Line; Chromatography, Thin Layer; Cisplatin; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Epidermal Growth Factor; Epithelium; Female; Humans; Lysophospholipids; MAP Kinase Signaling System; Neovascularization, Pathologic; Ovarian Neoplasms; Ovary; Pancreas; Phospholipase D; Phospholipases A; Phospholipases A2; Platelet-Derived Growth Factor; RNA; RNA, Ribosomal, 18S; Tumor Cells, Cultured

Greater than 95% of ovarian cancers originate in the epithelial cells on the surface of the ovary. The current study investigates the expression and action of transforming growth factor alpha (TGFalpha) in ovarian surface epithelium (OSE) and the underlying stroma in both normal and tumorigenic ovarian tissues. Normal bovine ovaries are used in the current study as a model system to investigate normal OSE functions. Transforming growth factor alpha and its receptor, the epidermal growth factor receptor (EGFR), were detected in the OSE from normal ovaries by immunocytochemistry (ICC). Ovarian stromal tissue also contained reduced but positive TGFalpha and EGFR immunostaining. To examine TGFalpha and EGFR gene expression, RNA was collected from normal bovine OSE and ovarian stromal cells. The TGFalpha and EGFR transcripts were detected in both fresh and cultured OSE and stromal cells by a sensitive quantitative reverse transcription polymerase chain reaction (QRT-PCR) assay. Transforming growth factor alpha gene expression was found to be high in freshly isolated OSE, but low in freshly isolated stroma. In contrast, EGFR expression was higher in the stroma compared to the OSE. Both the ICC and QRT-PCR indicate that normal OSE express high levels of TGFalpha in vivo and in vitro. In vitro, normal ovarian stromal cells develop the capacity to express high levels of EGFR. Human ovarian tumors from stage II, stage III, and stage IV ovarian cancer cases were found to express TGFalpha and EGFR protein in the epithelial cell component of the tumor by ICC analysis. The stromal cell component of human ovarian tumors contained little or no TGFalpha/EGFR immunostaining. Observations suggest that tumor progression may in part require autocrine stimulation of the epithelia. Transforming growth factor alpha was found to stimulate the growth of normal bovine OSE and stroma cells to the same level as epidermal growth factor. Two ovarian cancer cell lines, SKOV3 and OCC1, were also stimulated to proliferate in response to TGFalpha. Transforming growth factor alpha was also found to stimulate the expression of two growth factors previously shown to be produced by OSE. Transforming growth factor alpha stimulates both kit ligand/stem cell factor and keratinocyte growth factor production by OSE. The effect of hormones on TGFalpha and EGFR expression by the OSE was also examined. Human chorionic gonadotropin stimulated TGFalpha expression, but not FSH. Both hCG and FSH stimulate

Topics: Animals; Cattle; Cell Division; Chorionic Gonadotropin; DNA; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Gene Expression; Humans; Immunohistochemistry; Ovarian Neoplasms; Ovary; Reverse Transcriptase Polymerase Chain Reaction; Stromal Cells; Transforming Growth Factor alpha; Tumor Cells, Cultured

Endothelin (ET)-1 is produced in ovarian carcinoma cells and is known to act through ET(A) receptors as an autocrine growth factor in vitro and in vivo. In OVCA 433 human ovarian carcinoma cells, ET-1 caused phosphorylation of the epidermal growth factor receptor (EGF-R) that was accompanied by phosphorylation of Shc and its recruitment complexed with Grb2. These findings suggested that an EGF-R/ras-dependent pathway may contribute to the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) 2 and mitogenic signaling induced by ET-1 in these cells. Specific inhibition of EGF-R kinase activity by tyrphostin AG1478 prevented ET-1-induced transactivation of the EGF-R, as well as Shc phosphorylation and recruitment with Grb2. Furthermore, ET-1-induced activation of Erk 2 was partially inhibited by tyrphostin AG1478. In accord with this finding, the mitogenic action of ET-1 in OVCA 433 cells was also significantly reduced by a concentration of tyrphostin AG1478 that abolished the growth response of EGF-stimulated cells. Inhibition of protein kinase C activity, which contributes to the proliferative action of ET-1 in OVCA 433 cells, had no effect on the activation of Erk 2 by ET-1, which suggests that this effect of protein kinase C does not involve ras-independent activation of Erk 2. Inhibition by wortmannin of PI3-kinase activity, which has been implicated in ET-1 and other G protein-coupled receptor (GPCR)-mediated signaling pathways, reduced Erk 2 activation by ET-1 but had no effect on ET-1-induced EGF-R and Shc phosphorylation. These findings indicate that ET-1-induced stimulation of Erk 2 phosphorylation, and mitogenic responses in OVCA 433 ovarian cancer cells are mediated in part by signaling pathways that are initiated by transactivation of the EGF-R.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Cell Division; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; GRB2 Adaptor Protein; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Ovarian Neoplasms; Peptides, Cyclic; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase C; Proteins; Quinazolines; Receptor, Endothelin A; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Transcriptional Activation; Tumor Cells, Cultured; Tyrosine; Tyrphostins

Overexpression of epidermal growth factor (EGF) receptor and HER2 (p185neu) may both contribute to the growth of human cancers. A humanized anti-HER2 monoclonal antibody (mAb) 4D5 and a human-mouse chimeric anti-EGF receptor mAb C225 are currently being investigated in clinical trials for their anti-tumor activities. In the present study, we have examined the effect of concurrent treatment of OVCA 420 human ovarian cancer cells with mAb C225 and mAb 4D5. Exposure of OVCA420 cells to saturating concentrations of C225 (20 nM) for 7 days resulted in 40-50% growth inhibition, and exposure to 20 nM mAb 4D5 also resulted in 30-40% growth inhibition. The growth inhibition of OVCA420 cells by mAb C225 or 4D5 was associated with an increased G1 cell population; an increased level of a cyclin-dependent kinase (CDK) inhibitor p27Kip1 with increased association of p27kip1 with CDK2, CDK4 and CDK6; and decreased activities of these CDKs. Combination treatment with concurrent exposure to mAbs C225 and 4D5 resulted in additive anti-proliferative effects on these cells, which was accompanied by enhanced G1 cell distribution, a greater increase in the levels of p27Kip1 and a greater decrease in the activities of CDK kinases. The anti-proliferative effects and related changes in cell cycle regulators induced by mAb 4D5, mAb C225 or the combination of the two mAbs could be reversed by concurrent exposure to exogenous EGF. Our data suggest the potential fruitful cooperation of anti-EGF receptor mAb and anti-HER2 mAb in the treatment of human cancers stimulated by EGF receptor and HER2 signals.

Topics: Animals; Antibodies, Monoclonal; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; G1 Phase; Growth Inhibitors; Humans; Mice; Microtubule-Associated Proteins; Ovarian Neoplasms; Receptor, ErbB-2; Recombinant Fusion Proteins; Tumor Cells, Cultured; Tumor Suppressor Proteins

Epithelial ovarian cancer (EOC) has a high mortality rate, which is due primarily to the fact that early clinical symptoms are vague and nonspecific; hence, this disease often goes undetected and untreated until in its advanced stages. Sensitive and reliable methods for detecting earlier stages of EOC are, therefore, urgently needed. Epidermal growth factor (EGF) is a ligand for EGF receptor (ErbB1); this receptor is the product of the c-erbB1 proto-oncogene. ErbB1 overexpression is common in human ovarian carcinoma-derived cell lines and tumors, in which overexpression is thought to play a critical role in tumor etiology and progression. Furthermore, ErbB1 overexpression is associated with disease recurrence and decreased patient survival. Recently, we have developed an acridinium-linked immunosorbent assay that detects a approximately 110-kDa soluble analogue of ErbB1, ie., sErbB1, in serum samples from healthy men and women (A. T. Baron, et al., J. Immunol. Methods, 219: 23-43, 1998). Here, we demonstrate that serum p110 sErbB1 levels are significantly lower in EOC patients with stage III or IV disease prior to (P < 0.0001) and shortly after (P < 0.0001) cytoreductive staging laparotomy than in healthy women of similar ages, whereas EGF levels are significantly higher than those of age-matched healthy women only in serum samples collected shortly after tumor debulking surgery (P < 0.0001). We observe that the preoperative serum sErbB1 concentration range of advanced stage EOC patients barely overlaps with the serum sErbB1 concentration range of healthy women. In addition, we show that serum sErbB1 and EGF levels changed temporally for some EOC patients who were surgically debulked of tumor and who provided a second serum sample during the course of combination chemotherapy. Finally, we observe a significant positive association between sErbB1 and EGF levels only in serum samples of EOC patients collected prior to cytoreductive surgery (correlation coefficient = 0.61968; P = 0.0027). These data suggest that epithelial ovarian tumors concomitantly affect serum sErbB1 and EGF levels. In conclusion, these data indicate that serum sErbB1 and EGF (postoperative only) levels are significantly different between EOC patients and healthy women and that altered and/or changing serum sErbB1 and EGF levels may provide important diagnostic and/or prognostic information useful for the management of patients with EOC.

Topics: Acridines; Adult; Aged; Biomarkers, Tumor; Carcinoma; Case-Control Studies; Chemotherapy, Adjuvant; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Immunosorbent Techniques; Laparotomy; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Prognosis; Proto-Oncogene Mas; Reproducibility of Results; Sensitivity and Specificity; Survival Rate; Tumor Cells, Cultured

The erbB-2/HER2 oncogene is overexpressed in a significant fraction of human carcinomas of the breast, ovary, and lung in a manner that correlates with poor prognosis. Although the encoded protein resembles several receptors for growth factors, no high affinity ligand of ErbB-2 has so far been fully characterized. However, several lines of evidence have raised the possibility that ErbB-2 can augment signal transduction initiated by binding of certain growth factors to their direct receptors. Here, we contrasted these two models of ErbB-2 function: First, examination of a large series of epidermal growth factor (EGF)-like ligands and neuregulins, including virus-encoded ligands as well as related motifs derived from the precursor of EGF, failed to detect interactions with ErbB-2 when this protein was singly expressed. Second, by using antibodies that block inter-ErbB interactions and cells devoid of surface ErbB-2, we learned that signaling by all ligands examined, except those derived from the precursor of EGF, was enhanced by the oncoprotein. These results imply that ErbB-2 evolved as a shared receptor subunit of all ErbB-specific growth factors. Thus, oncogenicity of ErbB-2 in human epithelia may not rely on the existence of a specific ligand but rather on its ability to act as a coreceptor for multiple stroma-derived growth factors.

Topics: Breast Neoplasms; Carcinoma; Epidermal Growth Factor; Female; Genes, erbB-2; Glycoproteins; Humans; Ligands; Lung Neoplasms; Nerve Growth Factors; Neuregulins; Ovarian Neoplasms; Receptor, ErbB-2; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Stromal Cells; Tumor Cells, Cultured

Expression of the RIalpha subunit of cAMP-dependent protein kinase type I is increased in human cancers in which an autocrine pathway for epidermal growth factor-related growth factors is activated. We have investigated the effect of sequence-specific inhibition of RIalpha gene expression on ovarian cancer cell growth. We report that RIalpha antisense treatment results in a reduction in RIalpha expression and protein kinase A type I, and inhibition of cell growth. The growth inhibition was accompanied by changes in cell morphology and appearance of apoptotic nuclei. In addition, EGF receptor, c-erbB-2 and c-erbB-3 levels were reduced, and the basal and EGF-stimulated mitogen-activated protein kinase activities were reduced. Protein kinase A type I and EGF receptor levels were also reduced in cells overexpressing EGF receptor antisense cDNA. These results suggest that the antisense depletion of RIalpha leads to blockade of both the serine-threonine kinase and the tyrosine kinase signaling pathways resulting in arrest of ovarian cancer cell growth.

Topics: Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cell Size; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; Cyclic AMP-Dependent Protein Kinases; Down-Regulation; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Isoenzymes; Oligonucleotides, Antisense; Ovarian Neoplasms; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptor, ErbB-3; RNA, Messenger; Signal Transduction; Transfection; Tumor Cells, Cultured

The epidermal growth factor (EGF) induces the rapid formation of dendritic processes and the dissociation of SK-OV-3 ovarian cancer cells. The SK-OV-3 cell line is characterized by over-expression of the c-erbB-2 oncogene product (p185(c-erbB-2)). To investigate the role of p185(c-erbB-2) in cell motility, a c-erbB-2-specific single-chain antibody was expressed in SK-OV-3 cells using a retroviral expression vector. Eight individual clones expressing the single chain antibody were isolated. These clones have prominent retention of the cell-surface p185(c-erbB-2). The EGF-induced morphologic changes and cell motility of the single-chain-antibody-expressing clones were strongly inhibited, as observed in cell dissociation and in transmigration experiments. However, the suppression of p185(c-erbB-2) does not inhibit the motility signal elicited by 12-O-tetradecanoyl-phorbol-13-acetate. These data indicate that the c-erbB-2 oncogene product is essential to mediate the motility signal of EGF to SK-OV-3 ovarian cancer cells. The enhancement of tumor-cell motility may be partially responsible for the unfavorable prognosis of ovarian cancer with over-expression of c-erbB-2.

Topics: Cell Movement; Epidermal Growth Factor; ErbB Receptors; Female; Hepatocyte Growth Factor; Humans; Ovarian Neoplasms; Receptor, ErbB-2; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

We investigated the effects of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) on migration, invasion and proteinase expression of gynecological cultured cancer cells (SKG-IIIb cervical squamous cell carcinoma, OMC-4 cervical adenocarcinoma, SNG-M endometrial adenocarcinoma and OMC-3 ovarian adenocarcinoma), and whether these growth factors affect thymidine phosphorylase/platelet-derived endothelial cell growth factor expression of tumor cells. Tumor cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were stimulated by 0.1-10 nM EGF and TGF-alpha in a concentration-dependent manner. The zymography of tumor-conditioned medium showed that the treatment of tumor cells with EGF and TGF-alpha resulted in the increase of type IV collagenases, stromelysin and urokinase-type plasminogen activator which was partly confirmed by immunoblot analysis. The expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor which has angiogenic activity, was also upregulated by these growth factors. These results suggest that EGF and TGF-alpha act as positive regulators on the invasion process of gynecological tumor cells which may be associated with their stimulatory action on the motility of tumor cells, the expression of proteinases secreted by tumor cells and the angiogenic phenotype.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Movement; Endometrial Neoplasms; Epidermal Growth Factor; Female; Genital Neoplasms, Female; Humans; Metalloendopeptidases; Neoplasm Invasiveness; Ovarian Neoplasms; Thymidine Phosphorylase; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Uterine Cervical Neoplasms

Aberrant expression or activity of the epidermal growth factor (EGF) receptor family of tyrosine kinases has been associated with tumor progression and an invasive phenotype. In this study, we utilized 4 ovarian cancer cell lines, OVCA 432, DOV 13, OVEA6 and OVCA 429, to determine the effects of EGF on the regulation of proteolytic enzymes and their inhibitors, cellular migration and in vitro invasion. Induction of urinary-type plasminogen activator (u-PA) activity and tissue inhibitor of matrix metalloproteinase (TIMP)-1 was observed in all 4 cell lines. OVCA 432 cells showed strong PAI-1 induction; however, the other 3 lines displayed substantial baseline PAI-1 expression that was not induced by EGF. EGF-dependent stimulation of migration and induction of matrix metalloproteinase (MMP)-9 (gelatinase B) was observed in OVEA6 and OVCA 429 cells only. Upon EGF receptor activation, DOV 13, OVEA6 and OVCA 429 cells were induced to invade through an artificial basement membrane (Matrigel); however, no invasion was detected in OVCA 432 cells. Cell lines displaying induction of migration and MMP-9 (OVEA6 and OVCA 429) demonstrated robust EGF-induced invasion (5- to 20-fold), and cell invasion was substantially reduced in the presence of anti-catalytic MMP-9 antibody. Addition of anti-catalytic u-PA antibody inhibited the modest (<2-fold) EGF-induced invasion in a cell line that did not express MMP-9 (DOV 13) and in OVEA6 cells that displayed the highest baseline u-PA activity. Together, our findings indicate that multiple proteinases are important in ovarian cell invasion and implicate EGF induction of MMP-9 and migration as key components of more aggressive ligand-induced invasion.

Topics: Chemotaxis; Collagen; Collagenases; Culture Media, Conditioned; Dose-Response Relationship, Drug; Enzyme Induction; Epidermal Growth Factor; Female; Gelatinases; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metalloendopeptidases; Neoplasm Invasiveness; Ovarian Neoplasms; Plasminogen Activator Inhibitor 1; Tissue Inhibitor of Metalloproteinase-1; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Effects of sex steroids (estradiol-17 beta, E2; progesterone, Prog) and growth factors (epidermal growth factor, EGF; transforming growth factor-alpha, TGF-alpha) on invasive activity and 5'-deoxy-5-fluorouridine (5'-dFUrd) sensitivity of ovarian adenocarcinoma OMC-3 cells were investigated. Tumor cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were inhibited by 10 microM Prog, but stimulated by 0.1-10 nM EGF and TGF-alpha in a concentration-dependent manner. E2 did not have any effect on tumor cell migration or invasion. The zymography of tumor conditioned medium showed that the treatment of OMC-3 cells with EGF and TGF-alpha resulted in increases of type IV collagenase, stromelysin and urokinase-type plasminogen activator (uPA). EGF and TGF-alpha up-regulated thymidine phosphorylase (dThdPase) expression of tumor cells and consequently enhanced the antiproliferative action of 5'-dFUrd, which is converted to 5-fluorouracil by dThdPase. E2 and Prog did not have significant effects on the expression of proteolytic enzymes and dThdPase, or on the 5'-dFUrd sensitivity of tumor cells. The inhibitory effect of Prog on tumor cell invasion may depend on its inhibitory action on the motility of tumor cells. These results suggest that EGF and TGF-alpha simultaneously up-regulate the potential of ovarian adenocarcinoma cells to invade extracellular matrices and their dThdPase expression, both of which are associated with the specific action of 5'-dFUrd selectively to kill tumor cells with high invasive and metastatic potential.

Topics: Basement Membrane; Cell Division; Cell Movement; Collagenases; Culture Media, Conditioned; Cystadenocarcinoma, Mucinous; Drug Resistance, Neoplasm; Enzyme Induction; Epidermal Growth Factor; ErbB Receptors; Estradiol; Female; Fibronectins; Floxuridine; Humans; Matrix Metalloproteinase 3; Neoplasm Invasiveness; Neoplasm Proteins; Ovarian Neoplasms; Prodrugs; Progesterone; Receptors, Estrogen; Receptors, Progesterone; Thymidine Phosphorylase; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The biologic significance of epidermal growth factor (EGF)-related proteins and EGF receptor (EGFR) in the development and progression of human ovarian carcinoma was studied in 7 ovarian cystadenomas, 6 mucinous tumors of low malignant potential (LMP), and 25 invasive adenocarcinomas by immunohistochemistry. Results were correlated with clinicopathologic features. We also examined immunoreactivity in five serous adenocarcinomas both before and after cisplatin chemotherapy. Amphiregulin (AR) expression was observed only in mucinous tumors (4 of 8 cystadenomas, 2 of 6 tumors of LMP, and 6 of 10 cystadenocarcinomas), but was not detected in the serous tumors or clear cell adenocarcinomas. EGF, cripto, and EGFR expression was significantly higher in mucinous cystadenocarcinomas than in mucinous cystadenomas or mucinous tumors of LMP. Three of five specimens obtained at a second operation after chemotherapy had more intense or diffuse immunostaining for transforming growth factor alpha (TGF-alpha) than the initial specimens did. Coexpression of more than two of the EGF-related proteins or EGFR significantly correlated with increased surgical stage in serous and clear cell carcinoma. AR expression seems to correlate with mucinous differentiation rather than with advanced stages of ovarian tumors. Our results indicate that expression of some EGF-related proteins is greater in certain subtypes of ovarian carcinomas than in their benign counterparts and that coexpression of these proteins is associated with advanced stage in serous and clear cell carcinoma. Increased TGF-alpha expression may also be related to ovarian tumor resistance to cisplatin chemotherapy.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Biomarkers, Tumor; Cystadenoma; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Middle Aged; Ovarian Neoplasms; Transforming Growth Factor alpha

Endothelin 1 (ET-1) is produced in ovarian cancer cell lines and has been shown to act through ET(A) receptors as an autocrine growth factor to promote tumor cell proliferation in vitro. In OVCA 433 cells, the efficacy of ET-1 as a stimulus of [3H]thymidine incorporation was equivalent to that of epidermal growth factor. ET-1 also stimulated the rapid expression of c-fos, an action mediated by ET(A) receptors. The mitogenic action of ET-1 was not mediated by a pertussis toxin-sensitive G protein. An analysis of the effects of inhibition and depletion of protein kinase C (PKC) on mitogenic responses demonstrated that PKC was necessary but not sufficient for maximal stimulation by ET-1. In quiescent OVCA 433 cells, ET-1-induced stimulation of [3H]thymidine incorporation was prevented by two structurally distinct inhibitors of tyrosine kinase, herbimycin A and genistein. These results indicate that both PKC and protein tyrosine kinase participate in ET-1-stimulated mitogenic signaling. ET-1 rapidly stimulated tyrosine phosphorylation of several cellular proteins, among which p125FAK and p42 mitogen-activated protein kinase were identified. The additivity between the potent mitogenic actions of ET-1 and epidermal growth factor is consistent with the independence of their signal transduction pathways in ovarian cancer cells. These findings also indicate that intracellular signaling between the ET(A) receptor and a yet unidentified tyrosine kinase is involved in the mitogenic response to ET-1.

Topics: Benzoquinones; Blotting, Northern; Calcium-Calmodulin-Dependent Protein Kinases; Cell Adhesion Molecules; Cell Division; DNA, Neoplasm; Endothelin-1; Enzyme Inhibitors; Epidermal Growth Factor; Female; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation; Genes, fos; Genistein; Humans; Immunoblotting; Indoles; Isoflavones; Lactams, Macrocyclic; Maleimides; Ovarian Neoplasms; Pertussis Toxin; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Quinones; Rifabutin; Signal Transduction; Tumor Cells, Cultured; Virulence Factors, Bordetella

Granulosa cell tumors spontaneously occur in approximately 10-25% of female (SWR x SWXJ-9) F1 mice. The present studies were designed to test whether tumor-bearing mice produce a distinct hormonal profile by which they could be identified and determine whether cultured tumor cells are responsive to hormones and growth factors that regulate normal granulosa cells. Samples of female mouse blood taken from age 3 to 10 weeks allowed estimation of serum FSH, 17beta-estradiol, and inhibin levels for normal mice and for mice destined to develop tumors. These studies indicated that FSH and 17beta-estradiol values differed between normal and tumor-bearing animals, but overlapped sufficiently that such values could not accurately predict the tumor-bearing state. Inhibin concentrations did differentiate normal from tumor-bearing animals in all cases. Increased levels of inhibin were observed coincident in time with visibly detectable tumors within the ovaries. Compared to inhibin synthesis in vivo, hormonal responsiveness in vitro was much more variable. Steroidogenesis was stimulated in all tumors by dibutyryl-cAMP and low-density lipoprotein (LDL). Some, but not all, tumors responded to IGF1, EGF, FSH, and hCG. In about one-half of the tumors tested, FSH could induce hCG or dibutyryl-cAMP responsiveness. IGF1 pretreatment consistently increased the responsiveness of tumor cells stimulated by dibutyryl-cAMP and LDL. Production of inhibin by isolated tumor cells was detectable and decreased by EGF or dibutyryl-cAMP treatments. We conclude that granulosa tumor cell secretion of inhibin may be under different control than secretion from normal granulosa cells and acts as an excellent marker for these tumors.

Topics: Animals; Biomarkers, Tumor; Bucladesine; Epidermal Growth Factor; Estradiol; Female; Follicle Stimulating Hormone; Granulosa Cell Tumor; Inhibins; Insulin-Like Growth Factor I; Lipoproteins, LDL; Male; Mice; Ovarian Neoplasms; Tumor Cells, Cultured

Ovarian cancer is the second most common malignancy of the female reproductive tract. Approximately 50% of ovarian cancers have elevated levels of epidermal growth factor receptor (EGFR). This overexpression is correlated with a poor prognosis for patient survival. Ovarian cancers also express a number of sex steroid receptors. The androgen receptor (AR) is the predominant sex steroid receptor and is expressed in over 80% of ovarian cancers. We investigated whether a relationship exists between EGFR and AR in ovarian cancer. Sixty serous cystadenocarcinomas were analyzed for their relative levels of EGFR and AR by Western blot analysis. Data were analyzed by Student's t test and linear regression analysis for statistical significance. More than 98% of the tumors expressed detectable levels of EGFR, while 65% of the tumors expressed detectable levels of AR. The levels of EGFR (mean +/- SEM) were found to be significantly (P < 0.01) higher in AR+ (516 +/- 15) than in AR- (304 +/- 57) tumors. EGFR levels significantly correlated to AR levels (r = 0.49, P < 0.001). These results demonstrate an association between EGFR and AR levels in ovarian cancer. Whether this association represents a causal or a casual relationship remains to be determined.

Topics: Adult; Aged; Aged, 80 and over; Cystadenocarcinoma, Serous; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Ovarian Neoplasms; Receptors, Androgen

Growth factor receptor-signaling pathways are potentially important targets for anticancer therapy. The interaction of anticancer agents with specific molecular targets can be identified by correlating target expression patterns with cytotoxicity patterns. We sought to identify new agents that target and inhibit the activity of the epidermal growth factor (EGF) receptor and of c-erbB2 (also called HER2 or neu), by correlating EGF receptor, transforming growth factor (TGF)-alpha (a ligand for EGF receptor), and c-erbB2 messenger RNA (mRNA) expression levels with the results of cytotoxicity assays of the 49000 compounds in the National Cancer Institute (NCI) drug screen database.. The levels of mRNAs were measured and used to generate a molecular target database for the 60 cell lines of the NCI anticancer drug screen. The computer analysis program, COMPARE, was used to search for cytotoxicity patterns in the NCI drug screen database that were highly correlated with EGF receptor, TGF-alpha, or c-erbB2 mRNA expression patterns. The putative EGF receptor-inhibiting compounds were tested for effects on basal tyrosine phosphorylation, in vitro EGF receptor tyrosine kinase activity, and EGF-dependent growth. Putative ErbB2-inhibiting compounds were tested for effects on antibody-induced ErbB2 tyrosine kinase activity.. EGF receptor mRNA and TGF-alpha mRNA levels were highest in cell lines derived from renal cancers, and c-erbB2 mRNA levels were highest in cells derived from breast, ovarian, and colon cancers. Twenty-five compounds with high correlation coefficients (for cytotoxicity and levels of the measured mRNAs) were tested as inhibitors of the EGF receptor or c-erbB2 signaling pathways; 14 compounds were identified as inhibitors of these pathways. The most potent compound, B4, inhibited autophosphorylation (which occurs following activation) of ErbB2 by 50% in whole cells at 7.7 microM.. Novel EGF receptor or c-erbB2 pathway inhibitors can be identified in the NCI drug screen by correlation of cytotoxicity patterns with EGF receptor or c-erbB2 mRNA expression levels.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Line; Cluster Analysis; Colonic Neoplasms; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Ovarian Neoplasms; Receptor, ErbB-2; RNA, Messenger; Structure-Activity Relationship; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

Receptors for luteinizing hormone-releasing hormone (LH-RH) are found in nearly 80% of human ovarian cancers. The chemotherapeutic agent doxorubicin can be linked to [D-lysine6]LH-RH to form a cytotoxic analogue (AN-152) that may have greater specificity for tumor cells. This study was conducted to investigate the effects of AN-152 on the growth of LH-RH receptor-positive OV-1063 human epithelial ovarian cancers.. Nude mice bearing human ovarian tumors, OV-1063 or UCI-107 (LH-RH receptor negative), were injected intraperitoneally with saline (control) or with equimolar doses of AN-152 or doxorubicin; experiments involving mice with OV-1063 tumors also included groups that were administered [D-lysine6]LH-RH either alone or in combination with doxorubicin. Tumor volume, weight, doubling time, and burden (i.e., tumor weight/body weight) as well as tumor apoptotic and mitotic indices were determined. The levels of receptors for LH-RH and epidermal growth factor (EGF) and their messenger RNAs were measured by use of radioreceptor and reverse transcription-polymerase chain reaction assays, respectively.. The growth of OV-1063 ovarian tumors in nude mice, as based on reduction in tumor volume, was inhibited significantly (all P<.05, two-sided) 4 weeks after treatment with AN-152, even at the lowest dose tested (413 nmol/20 g weight); the toxic effects of an equivalent dose of doxorubicin caused substantial mortality. High-affinity receptors for LH-RH and EGF were found on cell membranes of OV-1063 cancers; however, after in vivo treatment with AN-152, LH-RH receptor-binding sites were not detectable and EGF receptors were reduced in number. The growth of UCI-107 ovarian cancers was not inhibited by AN-152.. In nude mice bearing LH-RH receptor positive OV-1063 epithelial ovarian cancers, systemic administration of AN-152 is less toxic and inhibits tumor growth better than equimolar doses of doxorubicin.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Southern; Carcinoma; Doxorubicin; Epidermal Growth Factor; ErbB Receptors; Female; Gonadotropin-Releasing Hormone; Humans; Luteinizing Hormone; Mice; Mice, Nude; Mitotic Index; Ovarian Neoplasms; Polymerase Chain Reaction; Receptors, LHRH; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Tumor Cells, Cultured

Many reports have cited coexpression of platelet-derived growth factor (PDGF) and its receptors by tumor cells or cells supporting tumor growth, suggesting both autocrine and paracrine mechanisms for PDGF-mediated tumor growth. We found that a small organic molecule, N-[4-(trifluoromethyl)phenyl] 5-methylisoxazole-4-carboxamide (SU101, leflunomide), inhibited PDGF-mediated signaling events, including receptor tyrosine phosphorylation, DNA synthesis, cell cycle progression, and cell proliferation. SU101 inhibited PDGF-stimulated tyrosine phosphorylation of PDGF receptor (PDGFR) beta in C6 (rat glioma) and NIH3T3 cells engineered to overexpress human PDGFRbeta (3T3-PDGFRbeta). SU101 blocked both PDGF- and epidermal growth factor (EGF)-stimulated DNA synthesis. Previously, this compound was shown to inhibit pyrimidine biosynthesis by interfering with the enzymatic activity of dihydroorotate dehydrogenase. In the current study, EGF-stimulated DNA synthesis was restored by the addition of saturating quantities of uridine, whereas PDGF-induced DNA synthesis was not, suggesting that the compound demonstrated some selectivity for the PDGFR pathway that was independent of pyrimidine biosynthesis. Selectivity was further demonstrated by the ability of the compound to block the entry of PDGF-stimulated cells into the S phase of the cell cycle, without affecting cell cycle progression of EGF-stimulated cells. In cell growth assays, SU101 selectively inhibited the growth of PDGFRbeta-expressing cell lines more efficiently than it inhibited the growth of PDGFRbeta-negative cell lines. SU101 inhibited the s.c., i.p., and intracerebral growth of a panel of cell lines including cells from glioma, ovarian, and prostate origin. In contrast, SU101 failed to inhibit the in vitro or s.c. growth of A431 and KB tumor cells, both of which express EGF receptor but not PDGFRbeta. SU101 also inhibited the growth of D1B and L1210 (murine leukemia) cells in syngeneic immunocompetent mice, without causing adverse effects on the immune response of the animals. In an i.p. model of tumor growth in syngeneic immunocompetent mice, SU101 prevented tumor growth and induced long-term survivors in animals implanted with 7TD1 (murine B-cell hybridoma) tumor cells. Because PDGFRbeta was detected on most of the tumor cell lines in which in vivo growth was inhibited by SU101, these data suggest that SU101 is an effective inhibitor of PDGF-driven tumor growth in vivo.

Topics: 3T3 Cells; Animals; Brain Neoplasms; Cell Survival; Epidermal Growth Factor; Female; Glioma; Growth Inhibitors; Humans; Isoxazoles; Leflunomide; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Ovarian Neoplasms; Platelet-Derived Growth Factor; Prostatic Neoplasms; Rats; Receptor, Platelet-Derived Growth Factor beta; Receptors, Platelet-Derived Growth Factor; Recombinant Proteins; Signal Transduction; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

The expression of mRNA for the epidermal growth factor (EGF) receptor, EGF and transforming growth factor alpha (TGF-alpha) was determined in 76 malignant, six borderline and 15 benign primary ovarian tumours using the reverse transcriptase-polymerase chain reaction and related to clinical and pathological parameters. Of the malignant tumours, 70% (53/76) expressed EGF receptor mRNA, 31% (23/75) expressed EGF mRNA and 35% (26/75) expressed TGF-alpha mRNA. For the borderline tumours, four of six (67%) expressed EGF receptor mRNA, 1/6 (17%) expressed TGF-alpha mRNA and none expressed EGF mRNA. Finally, 33% (5/15) of the benign tumours expressed EGF receptor mRNA, whereas 40% (6/15) expressed EGF mRNA and 7% (1/15) expressed TGF-alpha mRNA. The presence of the EGF receptor in malignant tumours was associated with that of TGF-alpha (P = 0.0015) but not with EGF (P = 1.00), whereas there was no relationship between the presence of EGF and TGF-alpha (P = 1.00). EGF receptor mRNA expression was significantly and positively associated with serous histology (P = 0.006) but not with stage or grade. Neither EGF nor TGF-alpha showed any link with histological subtype or stage. The survival of patients with malignant tumours possessing EGF receptor mRNA was significantly reduced compared with that of patients whose tumours were negative (P = 0.030 for all malignant tumours; P = 0.007 for malignant epithelial tumours only). In contrast, neither the expression of TGF-alpha nor EGF was related to survival. These data suggest that the presence of EGF receptor mRNA is associated with poor prognosis in primary ovarian cancer.

Topics: Base Sequence; Carcinoma; DNA Primers; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Multivariate Analysis; Ovarian Neoplasms; Prognosis; RNA, Messenger; RNA, Neoplasm; Survival Analysis; Teratoma; Tumor Necrosis Factor-alpha

Alterations in the expression of the epidermal growth factor (EGF) receptor ErbB family are frequently encountered in a number of human cancers. Two of these receptors, ErbB3 and ErbB4, are known to bind a family of related proteins termed heregulins (HRGs) or neu differentiation factors. In biologically relevant systems, interaction of HRG with ErbB3 or ErbB4 results in the transactivation of ErbB2. In this report, we show that ErbB2 is a critical component in mediating HRG responsiveness in a panel of human breast and ovarian tumor cell lines. Because HRGs have been reported to elicit diverse biological effects on cultured cells, including growth stimulation, growth inhibition, and induction of differentiation, we systematically examined the effect of rHRG beta 1 on tumor cell proliferation. HRG binding studies were performed with a panel of breast and ovarian tumor cell lines expressing a range of levels of ErbB2. The biological responses to HRG were also compared to EGF and to the growth-inhibitory anti-ErbB2 antibody, 4D5. In most cases, HRG stimulation of DNA synthesis correlated with positive effects on cell cycle progression and cell number and with enhancement of colony formation in soft agar. On each cell line tested, the HRG effects were distinguishable from EGF and 4D5. Our findings indicate that HRG induces cell proliferation in a number of tumor cell lines. In addition, we show that methods for measuring cell proliferation, as well as experimental conditions, are critical for determining HRGs effect on tumor cell growth in vitro.

Topics: Animals; Antibodies, Monoclonal; Breast Neoplasms; Carrier Proteins; Cell Adhesion; Cell Count; Cell Cycle; Cell Division; Epidermal Growth Factor; ErbB Receptors; Female; Glycoproteins; Humans; Neuregulin-1; Ovarian Neoplasms; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptor, ErbB-3; Tumor Cells, Cultured

The effects of EGF and TGF beta 1 on onco gene expressions was studied by RT-PCR technique in an ovarian cancer cell line HO-8910. The results showed that TGF beta 1 could inhibit mRNA expression of TGF beta 1 gene and that of c-myc, EGFR and c-erbB2 genes in HO-8910 cells in vitro. However, EGF could enhance the mRNA expressions of c-myc, c-erbB2 and EGFR to various extents, but inhibit that of TGF beta 1 gene.

Topics: Cystadenocarcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Genes, erbB-2; Genes, myc; Humans; Ovarian Neoplasms; Recombinant Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

To investigate the role of growth factors in growth regulation of human ovarian cancer cells by in vitro.. The regulation effects of different growth factors on human ovarian cancer cell line HO-8910, such as on cell proliferation, DNA synthesis, the alteration of distributions of cell phase fractions and the changes of cyclic adenosine monophosphate (cAMP) level in the presence of 2.5% fetal bovine serum (FBS) were analyzed.. (1) Insulin-like growth factor, transforming growth factor-alpha (TGF-alpha) showed weak and positive effects and were dose dependent. Epidermal growth factor (EGF) strongly stimulated cell growth and DNA synthesis in HO-8910 cells, in contrast, TGF-beta 1 inhibited cell proliferation and DNA synthesis. (2) Following the actions of various growth factors on cells in vitro, corresponding alterations in the distributions of cell phase fractions, in the formation of silver-staining nucleolar organizer regions (Ag-NORs) and in the cAMP content appeared.. The growth factors exert their growth regulating effects on ovarian cancer cells through intracellular signal transduction to bring about changes in nuclear DNA synthesis and alterations in the distribution of cell phase fractions. In the process of growth regulation on ovarian cancer cells by growth factors, the change of cAMP content presents double direction regulatory function. The growth inhibitory regulation of the TGF-beta 1 possibly functions as an autocrine. This report presents evidence supporting the important roles of the growth factors and may further the study into the mechanism of growth regulation by growth factors in this cell line.

Topics: Cell Cycle; Cell Division; Cystadenocarcinoma, Serous; DNA, Neoplasm; Epidermal Growth Factor; Female; Humans; Ovarian Neoplasms; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Growth factors have been demonstrated to regulate the proliferation and viability of a number of cell lineages. Because most drugs used in chemotherapy kill cells through programmed cell death, by the process of apoptosis, we determined whether growth factors, specifically epidermal growth factor (EGF) and lysophosphatidic acid (LPA), which we have demonstrated recently to be a potent growth factor for ovarian cancer cells, would alter the ability of cis-diamminedichloroplatinum (cis-DDP), the most effective chemotherapeutic agent for ovarian cancer, to kill the HEY ovarian cancer cell line. We demonstrate that both EGF and LPA decrease the ability of cis-DDP to kill HEY ovarian cancer cells as assessed by colony-forming cell activity and dye reduction. Morphological changes, DNA release, and electron microscopy suggested that LPA and EGF protect ovarian cancer cells from programmed cell death induced by cis-DDP. Because LPA is present in high levels in ascitic fluid from ovarian cancer patients, and the EGF receptor is expressed by tumor cells from a significant portion of patients where it correlates with prognosis, growth factor modulation of cis-DDP-induced apoptosis may play a role in the poor prognosis associated with ovarian cancer.

Topics: Antineoplastic Agents; Apoptosis; Cell Survival; Cisplatin; DNA Fragmentation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lysophospholipids; Ovarian Neoplasms

Despite significant infiltration into tumors and atherosclerotic plaques, the role of T lymphocytes in these pathological conditions is still unclear. We have demonstrated that tumor-infiltrating lymphocytes (TILs) and plaque-infiltrating lymphocytes (PILs) produce heparin-binding epidermal growth factor-like growth factor (HB-EGF) and basic fibroblast growth factor (bFGF) in vitro under nonspecific conditions and in vivo in tumors by immunohistochemical staining. HB-EGF and bFGF derived from TILs and PILs directly stimulated tumor cells and vascular smooth muscle cells (SMCs) in vitro, respectively, while bFGF displayed angiogenic properties. Therefore, T cells may play a critical role in the SMC hyperplasia of atherosclerosis and support tumor progression by direct stimulation and angiogenesis.

Topics: 3T3 Cells; Animals; Arteriosclerosis; Biological Assay; Breast Neoplasms; Cell Division; Chromatography, Affinity; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lymphocytes, Tumor-Infiltrating; Mice; Ovarian Neoplasms; T-Lymphocytes

In order to investigate the biological significance of epidermal growth factor (EGF) in the cell function of teratocarcinoma cells, we examined the production, binding and cell proliferative effect of EGF in PA-1 human ovarian teratocarcinoma cell line. The immunoreactivity of EGF in PA-1 cell-conditioned medium was detected by human EGF radioimmunoassay, and prepro-EGF mRNA was demonstrated in PA-1 cells by Northern blot analysis. An [125I]EGF binding study showed the presence of EGF receptor with very high binding affinity and relatively low numbers of binding sites in PA-1 cells. Furthermore, the growth of PA-1 cells was stimulated by EGF and inhibited by anti-EGF monoclonal antibody. These results suggest strongly that EGF plays an important role in controlling the growth of teratocarcinoma cells as an autocrine/paracrine growth factor.

Topics: Blotting, Northern; Cell Division; Cell Line; Culture Media, Conditioned; Epidermal Growth Factor; Female; Humans; Ovarian Neoplasms; Radioligand Assay; RNA, Messenger; Teratocarcinoma

mRNA amplification phenotyping (MAPPing) was used to determine the level of mRNA expression of the major EGF-related ligands (EGF, TGF-alpha, and Amphiregulin) and receptors (EGF-receptor and erbB-2) of the EGF supergene family in three ovarian carcinoma lines (OVCA 429 and 433, and NIH:OVCAR-8) under serum-supplemented and reduced serum (minimal medium with 2% fetuin) growth conditions. mRNA levels of TGF-alpha, EGF-R, and erbB-2 were particularly high, and increased approximately 2-3 orders of magnitude when grown in serum, consistent with an autocrine involvement of these genes in ovarian epithelial growth in vitro. Moreover, even when grown without serum, OVCA 429 and NIH:OVCAR-8 expressed elevated levels of mRNA for erbB-2.

Topics: Amphiregulin; Base Sequence; DNA, Neoplasm; EGF Family of Proteins; Epidermal Growth Factor; Epithelium; ErbB Receptors; Female; Gene Amplification; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Molecular Sequence Data; Ovarian Neoplasms; Phenotype; Polymerase Chain Reaction; Receptor, ErbB-2; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

Primary and metastatic ovarian cystadenocarcinomas, carcinomas of low malignant potential (borderline tumors), benign ovarian cystadenomas, and normal ovaries were compared for immunoperoxidase detection of the ligands epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), amphiregulin (AR), cripto, and the receptors, epidermal growth factor receptor (EGF-R), and c-erbB-2. This matrix analysis of these EGF family members indicated no specific pattern of ligand or receptor expression with a specific ovarian histologic category except in the case of AR and TGF-alpha. AR was detected almost exclusively in borderline tumors, suggesting that these tumors may not arise as a pathological continuum between benign cystadenomas and invasive cystadenocarcinomas. Second, the presence of TGF-alpha immunoreactivity in the absence of coexpression of cripto or EGF appeared to be associated only with adenocarcinomas of high grade and stage.

Topics: Amphiregulin; Cell Transformation, Neoplastic; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Female; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Multigene Family; Neoplasm Proteins; Ovarian Neoplasms; Receptor, ErbB-2; Transforming Growth Factor alpha

Circulating epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) were estimated in 58 patients with epithelial ovarian cancer and were correlated with clinically and biochemically important prognosticators. IGF-I levels were significantly low in patients as compared to controls. The relation of growth factors with clinically important prognosticators was non-significant. Moreover, the levels of EGF and IGF-I in the ER+/PR+ and ER-/PR- groups and in the low and high EGFR+ tumors did not differ significantly. Patients with EGF < 1.0 ng/ml had significantly better survival than those with EGF > 1.0 ng/ml.

Topics: Epidermal Growth Factor; Epithelium; Female; Humans; Insulin-Like Growth Factor I; Neoplasm Staging; Ovarian Neoplasms; Prognosis; Receptors, Estrogen; Receptors, Progesterone; Reference Values

The levels of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) were analysed in 24-h urine samples from patients with ovarian malignancies, benign ovarian tumours, and healthy controls by specific radioimmunoassays. No significant difference in total urinary immunoreactive EGF excretion between the groups was detected. However, 79% (23/29) of the patients with ovarian carcinomas excreted TGF-alpha (median 12.6 pmol/24 h), whereas only 17% (2/12) of the patients with benign ovarian tumours and 23% (3/13) of the controls did so. The difference between cancer patients and controls was highly significant (P < 0.001). Analyses of the urine samples separated by gel filtration revealed a greater molecular heterogeneity of EGF and TGF-alpha in cancer patients than in controls. High and low molecular weight forms of EGF were able to bind to the EGF receptor and to induce anchorage-independent growth. After surgical reduction of the tumour, a distinct decrease of urinary high molecular weight forms was observed. Thus, some macromolecular growth factors seem to be associated with epithelial ovarian carcinomas.

Topics: Biomarkers, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Molecular Weight; Ovarian Neoplasms; Postoperative Period; Radioimmunoassay; Radioligand Assay; Transforming Growth Factor alpha

The importance of epidermal growth factor (EGF) receptor-dependent growth has not been clarified for in vivo growth of primary human ovarian cancers.. Seventeen primary human ovarian cancer tissue samples were examined for the presence of EGF receptors by a 125I-EGF-binding study. Three groups of mice were inoculated with EGF receptor expressing and not-expressing cancer tissues. The groups were as follows: control group, Sx group (mice that underwent sialoadenectomy; EGF depleted mice), and Sx+EGF (EGF-replaced) group. The ability of the inoculated tissues to implant and grow then was studied.. Of the 17 primary ovarian cancers, 12 expressed EGF receptors and 5 did not. Eight of 12 EGF-receptor expressing cancer tissues implanted and formed growing tumors in control animals. None implanted in the Sx animals. Epidermal growth factor receptor-expressing cancers implanted in Sx animals that received EGF administration. Two of five EGF receptor-negative ovarian cancers implanted and grew in both control and Sx animals.. Growth of EGF receptor-expressing primary human ovarian cancers may be dependent on EGF in vivo.

Topics: Animals; Base Sequence; Cell Membrane; Cystadenocarcinoma, Mucinous; Cystadenocarcinoma, Serous; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Iodine Radioisotopes; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Transplantation; Ovarian Neoplasms; Polymerase Chain Reaction; Protein Binding; RNA, Neoplasm; Salivary Glands; Transcription, Genetic; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured

Reverse transcription polymerase chain reaction (RT-PCR) revealed that the Fallopian tubes express epidermal growth factor (EGF), transforming growth factor (TGF alpha), and EGF receptor (EGF-R) mRNA. The RT-PCR product was verified by restriction enzyme digestion analysis. Immunohistochemically, EGF, TGF alpha, and EGF-R were localized in Fallopian tubes by use of specific antibodies to human EGF, mature fragments of human TGF alpha, and monoclonal antibodies to the extracellular binding domain of EGF-R. The tubal epithelial cells were the primary site of immunoreactive EGF, TGF alpha, and EGF-R, which were present to a lesser extent in the stromal cells, smooth muscle cell layers, fibroblasts of serosal tissue, and arterial endothelial and smooth muscle cells. Using antibodies generated against the amino and carboxy termini of TGF alpha precursor produced a similar cellular distribution to that observed for mature TGF alpha. The intensity of immunoreactive TGF alpha with these antibodies was similar to that seen with EGF. The ciliated and nonciliated epithelial cells in the ampullary and isthmus regions immunostained with similar intensity for EGF, TGF alpha, and EGF-R. The immunostaining for EGF, TGF alpha, and EGF-R was cycle-dependent, was considerably higher during late proliferative and early-to-mid-secretory phases than during early proliferative and late secretory phases of the menstrual cycle, and was reduced during the postmenopausal period. Specimens obtained 5-12 yr after tubal ligation immunostained for EGF, TGF alpha, and EGF-R similarly to sections from unligated tubes taken during the same phase of the cycle. Quantitative autoradiography of 125I-EGF binding generated a pattern similar to that of immunostaining for EGF-R binding. Net grain density/100 microns 2 calculated for different cell types indicated that the epithelial cells had a significantly higher grain density than did other tubal cell types (p < 0.05) without the cycle dependency seen in the immunohistochemical study. In summary, the results demonstrate that the human Fallopian tube expresses mRNA and contains immunoreactive proteins for EGF, TGF alpha, and EGF-R as well as binding sites for 125I-EGF. The cycle dependency and lower immunostaining in postmenopausal tubes suggest a potential regulation of their expression by ovarian steroids. The results imply the importance of EGF/TGF alpha in a variety of tubal biochemical and physiological functions and possibly early embryon

Topics: Autoradiography; DNA Restriction Enzymes; Endothelium, Vascular; Epidermal Growth Factor; ErbB Receptors; Fallopian Tubes; Female; Gene Expression; Humans; Immunohistochemistry; Male; Muscle, Smooth; Ovarian Neoplasms; Polymerase Chain Reaction; Pregnancy; RNA, Messenger; Transforming Growth Factor alpha; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

In this lecture, importance of growth factors in reproductive functions and cancer growth are discussed. Among many kinds of growth factors, epidermal growth factor (EGF) and transforming growth factor (TGF)alpha are mentioned; both of these are called as EGF family because these share a common receptor (EGF receptor) and show similar biological activities. It is known that growth factors are important in reproductive fields. They play vital roles in the follicle development and the endometrial proliferation in response to estrogen. We studied the expression and role of EGF and TGF alpha in human fallopian tube. Immunohistochemical and RT-PCR studies revealed the menstrual stages specific synthesis and expression of EGF and TGF alpha in tubal epithelial cells. They were abundant at the late follicular and luteal stages, were little at the early follicular stage, suggesting that these growth factors are expressed in response to estrogen. We, next, examined the role of these factors in early embryo development using mouse embryos. Embryo development was significantly improved when embryos were co-cultured with human tubal epithelial cells. However, the favorable effects of the tubal epithelial cells were almost completely abolished by the addition of anti-EGF and -TGF alpha neutralizing antibodies. These facts suggest that EGF and EGF alpha expressed in human epithelial cells promote embryo development in a paracrine manner. Autocrine mechanisms by growth factors are known to be important on cancer cell growth. Among many kinds of autocrine mechanisms TGF alpha/EGF receptor autocrine mechanism is the most commonly expressed in many kinds of cancers such as lung cancer, esophageal cancer, pancreas cancer and liver cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Embryonic and Fetal Development; Epidermal Growth Factor; Female; Humans; Mice; Ovarian Neoplasms; Transforming Growth Factor alpha

A series of rat monoclonal antibodies (MAbs) has been generated against the extracellular domain of the receptor for EGF which block the binding of EGF and TGF alpha to the receptor and inhibit the growth in vitro of a range of carcinoma cell lines that over-express the receptor for EGF. Some of these antibodies were able also to induce the complete regression of xenografts of EGFR-over-expressing tumours when treatment was started, either at the time of tumour inoculation or later when the tumours were established. The most effective of these antibodies was ICR62, which was also able to activate host immune effector functions. We conclude that antibodies which block growth-factor-ligand interaction can have a profound influence on the proliferative capacity of tumour cells in vivo and may have useful clinical application.

Topics: Animals; Antibodies, Monoclonal; Binding Sites; Breast Neoplasms; Carcinoma; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Head and Neck Neoplasms; Humans; Immunotherapy; Lung Neoplasms; Mice; Mice, Nude; Ovarian Neoplasms; Rats; Rats, Inbred Strains; Recombinant Proteins; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vulvar Neoplasms

Cluster 13 was defined by 2 independently derived murine monoclonal antibodies (MAbs), RS7 (IgG1) and MR54 (IgG2a), which were raised against human squamous-cell carcinoma of the lung and a human ovarian-carcinoma cell line, respectively. Immunologic and biochemical evidence demonstrated that RS7 and MR54, as well as 2 additional MAbs, MR6 (IgG2a) and MR23 (IgG1), generated in the same fusion as MR54, recognize the same antigen, a 46- to 48-kDa glycoprotein. Evaluation of the expression of antigen on the surface of tumor cell lines, Western blotting analyses, competitive binding studies, and double-determinant ELISA assays, support this conclusion. Two distinct epitopes are defined by these MAbs. In order to further characterize this antigen, amino-acid-sequence analyses were performed on peptides derived from antigen purified by affinity chromatography with MAb RS7. The sequence data obtained from 2 peptides, which were independently generated by CNBr cleavage and trypsin digestion respectively indicated identity to GA733-1. The GA733-1 genomic DNA sequence predicted a type-1 membrane protein of 35 kDa, with 4 potential N-linked glycosylation sites. The GA733-1 protein product has not been identified previously, and MAbs to this tumor-associated antigen were not previously known.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Blotting, Western; Carcinoma, Squamous Cell; Cell Adhesion Molecules; Cell Line; Epidermal Growth Factor; Epithelial Cell Adhesion Molecule; Female; Humans; Immunoglobulin G; Lung Neoplasms; Mice; Molecular Sequence Data; Multigene Family; Neoplasms; Ovarian Neoplasms; Sequence Homology, Amino Acid; Tumor Cells, Cultured

The factors controlling cell growth in epithelial ovarian cancer (OV Ca) remain poorly defined. Epidemiological evidence suggests that gonadotropins and estrogens may be important in the development of OV Ca. We have established permanent cultures of OV Ca cells from a mesenteric metastasis of an ovarian papillary adenocarcinoma. The growth of these cells was increased 20-60% (p < 0.01) by picomolar concentrations of 17 beta-E2 (but not by 17 alpha-E2) and 0.1 to 1 microgram/mL of hCG (but not hFSH). Both EGF and IGF-1 increased cell growth, and a maximal effect of 3- to 5-fold increase in cell number was observed. Under conditions in which the growth responses to EGF and IGF-1 were submaximal, E2 (0.1 to 100 nM) produced a concentration related increase in the growth response to IGF-1 and EGF. Ten nM E2 increased the responses to EGF and IGF-1 more than 2-fold. In combination, E2/hCG enhanced the growth response to EGF but not to IGF-1. Estradiol increased the Ka of the EGF receptor approximately 2-fold (p < 0.01) and increased the number of receptors/cell for IGF-1 approximately 50% (p < 0.01). While hCG had no effect on EGF receptor binding parameters, it increased the Ka of the IGF-1 receptor and enhanced the effect of E2 on IGF-1 receptor number. These studies suggest that E2 and hCG may regulate cell growth responses of OV Ca cells to IGF-1 and EGF. These changes may be exerted at least partly through modulation of properties of receptors for the two growth factors.

Topics: Adenocarcinoma, Papillary; Aged; Cell Division; Chorionic Gonadotropin; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Estradiol; Female; Humans; Insulin-Like Growth Factor I; Ovarian Neoplasms; Receptor, IGF Type 1; Tumor Cells, Cultured

Epidermal growth factor (EGF) elicits estrogen receptor (ER)-dependent physiological sequelae and estrogen-like biochemical effects on the ER in the mouse uterus. These in vivo observations indicate that EGF may elicit some of its actions by activation of the ER. The effect of peptide growth factors on activation of a consensus estrogen-responsive element was assessed in a strain of Ishikawa human endometrial adenocarcinoma cells with negligible levels of ERs, as determined by Western blot and [3H]estradiol binding, and in BG-1 human ovarian adenocarcinoma cells, which contain abundant ERs. EGF and transforming growth factor-alpha induced transcriptional activation of a consensus ERE in an ER-dependent manner in both cell types. Transcriptional activation by the growth factors was inhibited by ICI 164,384, an ER receptor antagonist, and neutralizing antibodies to the EGF receptor. Immunodetection of the ER in BG-1 cells demonstrated that receptor levels were not induced by transforming growth factor-alpha vs. untreated cells. ER deletion mutants containing amino acids 1-339 and 121-599 were transfected into Ishikawa cells. The 1-339 mutant was more active in inducing transcription after EGF treatment than the 121-599 mutant. Estrogen only stimulated transcription in the presence of the 121-599 mutant, while 1-339 was inactive. Interestingly, synergism between a physiological dose of estrogen and peptide growth factors was observed. The presence of cross-talk between EGF receptor and ER signaling pathways suggests that interactions between growth factors and steroid receptors may modulate hormonal activity influencing normal and aberrant function in mammalian cells.

Topics: Adenocarcinoma; Animals; Base Sequence; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Female; Gene Expression Regulation; Humans; Mice; Molecular Sequence Data; Ovarian Neoplasms; Polyunsaturated Alkamides; Promoter Regions, Genetic; Receptors, Estrogen; Recombinant Fusion Proteins; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Signal Transduction; Simian virus 40; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured; Uterine Neoplasms; Vitellogenins

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) content was measured in normal ovaries and benign ovarian tumours. Epidermal growth factor was present in 12.7% of normal ovaries, with a range 0.030-0.533 ng/mg DNA, and in 31.8% of benign ovarian tumours, with a range 0.1335-2.080 ng/ml DNA. TGF alpha was present in 84.5% of normal ovaries, with a range of values from 0.037-18.2 ng/mg DNA, and in 84.1% of benign ovarian tumours, with a range of 0.083-195 ng/mg DNA. The TGF alpha content in post menopausal benign ovarian tumours was significantly higher (P < 0.0001) than TGF alpha in the pre-menopausal normal ovarian group. The frequency of detection and levels of TGF alpha measured were significantly higher than those of EGF in the normal ovary group (P < 0.001) and also in the benign ovarian group (P < 0.005). We conclude that TGF alpha is the predominant growth factor present in normal ovaries and benign ovarian tumours.

Topics: Adult; Aged; Epidermal Growth Factor; Female; Humans; Middle Aged; Ovarian Neoplasms; Ovary; Transforming Growth Factor alpha

Indirect evidence suggests that gonadal steroids and gonadotropins may have a role in the genesis of epithelial ovarian cancer. In the studies reported herein, we established 17 beta-estradiol (E2) secreting cell cultures from an omental metastasis of an epithelial ovarian cancer. We demonstrate that human chorionic gonadotropin (hCG), human follicle-stimulating hormone, and epidermal growth factor (EGF) increased cell growth in a dose- and time-dependent manner, whereas E2 inhibited cell growth in the nanomolar range. Epidermal growth factor was able to partially block the negative effect of E2; a similar but quantitatively lesser effect was observed with hCG. These results provide direct evidence to support the view that gonadotropins, EGF, TGF beta (transforming growth factor), and estradiol may modulate growth of metastatic epithelial ovarian cancer cells.

Topics: Cell Count; Cell Division; Chorionic Gonadotropin; Cyclic AMP; Cystadenocarcinoma; Dose-Response Relationship, Drug; Drug Interactions; Epidermal Growth Factor; Estradiol; Female; Follicle Stimulating Hormone; Humans; Middle Aged; Omentum; Ovarian Neoplasms; Peritoneal Neoplasms; Time Factors; Tumor Cells, Cultured

The effects of EGF and TGF-beta 1 on the proliferation of 2 ovarian carcinoma cell lines (IGROV1 and OVCCR1) were evaluated. The cell lines were adapted to grow in a restricted serum (0.5%) medium. EGF was required for proliferation of both ovarian cell lines. Low doses of TGF-beta 1 inhibited clonogenic capacity and attenuated the EGF-mediated stimulation of DNA synthesis in OVCCR1 cells. TGF-beta 1 inhibited OVCCR1 cell proliferation by blocking the cell cycle at the G1/S transition. TGF-beta 1 did not affect either clonal or monolayer growth of IGROV1 cells. Both cell lines express type-I and type-III TGF-beta receptors, suggesting that the unresponsiveness of IGROV1 cells to TGF-beta 1 occurs at a post-receptor level. TGF-beta 1 mRNA was detected in OVCCR1 cells and in 8 out of 11 of the ovarian tumor specimens examined.

Topics: Cell Division; DNA; Epidermal Growth Factor; Female; Gene Expression; Growth Inhibitors; Humans; Ovarian Neoplasms; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tumor Cells, Cultured

Over-expression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian, endometrial, and mammary carcinoma is an indicator of poor prognosis. Interactions between the epidermal growth factor (EGF) receptor and the HER-2 protein have been described. The aim of this study was to elucidate the effects of EGF on HER-2 expression. In the human ovarian carcinoma cell lines HTB-77, OVCAR-3, 2780, SKOV-6, SKOV-8 and 2774, and the human mammary tumor cell line SKBR-3, total cellular p185HER-2 was determined by an ELISA, whereas the surface p185HER-2 was measured with a living-cell RIA. Stimulation of these cell lines with either EGF (0.1-30 nM) or TGF-alpha (0.1-30 nM) led to a significant reduction in p185HER-2 expression. The effect was more pronounced in cells with normal HER-2 expression. A reduction of mRNA levels for p185HER-2 by EGF was observed in OVCAR-3 cells but not in the over-expressing lines HTB-77 and SKBR-3. Interestingly, the EGF-induced effect was not always associated with growth stimulation and was not correlated with the number of EGF binding sites detected by a radioligand assay. Our data indicate that EGF treatment results in a down-regulation of p185HER-2.

Topics: Base Sequence; Breast Neoplasms; Cell Division; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Molecular Sequence Data; Ovarian Neoplasms; Proto-Oncogene Mas; Proto-Oncogene Proteins; Receptor, ErbB-2; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

We studied the correlation between dexamethasone (Dex) induced growth effects and modulation of epidermal growth factor receptor (EGFR) expression in OVCA 433 ovarian cancer cells. These cells express specific high and low affinity 125I-EGF binding sites and are growth stimulated by EGF. Dex exhibits mitoinhibitory effects by recruiting OVCA 433 cells in the G0-G1 phase of the cycle, but increases the number of both the high and the low affinity EGFR in a dose dependent manner. The maximal EGFR expression increase occurs after 24 h of Dex treatment consistently with Northern blot studies. The mitogenic activity of EGF in OVCA 433 cells is not affected by the presence of Dex. Moreover Dex growth inhibition occurs in JA1 cells, an ovarian cancer cell line which expresses unfunctional EGFR and which is unresponsive to EGF. Our results indicate that the Dex induced growth effects occur independently of EGFR expression.

Topics: Antibodies, Monoclonal; Binding Sites; Blotting, Northern; Cell Division; Dexamethasone; Dose-Response Relationship, Drug; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Interphase; Ovarian Neoplasms; RNA; Tumor Cells, Cultured

Regulation of ovarian cancer growth is poorly understood. In this study, the effects of EGF, TGF alpha and TGF beta 1 on two ovarian cancer cell lines (OVCAR-3 and CAOV-3) were investigated. The results showed that EGF/TGF alpha stimulated cell growth and DNA synthesis in OVCAR-3 cells, but inhibited cell proliferation and DNA synthesis in CAOV-3 cells. TGF beta 1 invariably inhibited cell proliferation and DNA synthesis in both cell lines. These effects on growth factors are dose dependent. The interaction of TGF beta 1 and EGF/TGF alpha was antagonistic in OVCAR-3 cells. In contrast, EGF/TGF alpha and TGF beta 1 had an additive inhibitory effect on CAOV-3 cells. Our results demonstrated that mature and functional EGF receptors are present in both cell lines and that they are capable of ligand binding, internalization, processing and ligand-enhanced autophosphorylation. Both high- and low-affinity binding are present in these cell lines, with CAOV-3 cells having about 2-3-fold higher total receptors than OVCAR-3 cells. These results together with those from our previous studies show that these cells express TGF alpha, TGF beta 1 and EGF receptors and that cell growth may be modulated by these growth factors in an autocrine and paracrine manner. This report presents evidence supporting the important roles of growth factors in ovarian cancer growth and provides a foundation for further study into the mechanism of growth regulation by growth factors in these cell lines.

Topics: Cell Division; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Phosphorylation; Thymidine; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Promotion of 'initiated' JB6 epidermal cells to the tumor phenotype can be effected by 12-O-tetradecanoylphorbol-13-acetate treatment, by stimulation of epidermal growth factor (EGF) receptor activity with EGF or transforming growth factor alpha and by exposure to the isoquinoline derivative H7. When these cells were incubated with pertussis toxin (PTX), induction of anchorage-independent growth by all four promoting substances was suppressed. The inhibition is specific since cell proliferation is not affected, suggesting that activation of a Gi protein is essential for promotion of the epidermal cells. This interpretation is strongly supported by the observation that the wasp poison mastoparan, which is known to mimic receptor-mediated activation of certain Gi proteins, also promoted anchorage independence. Immunological data and partial amino acid sequence analysis of ADP-ribosyl alpha i isolated from PTX-treated JB6 cells indicate that a Gi-2 protein is a mediator to tumor promotion in this system. The inhibitory action of 4-bromophenacyl bromide may point to a coupling of the Gi protein to phospholipase A2. From our data we infer that promoters induce the tumor phenotype in 'initiated' JB6 epidermal cells by activating epigenetically the same Gi protein that in a number of adrenal and ovarian tumors appears to be persistently activated by mutational events.

Topics: Adrenal Gland Neoplasms; Amino Acid Sequence; Animals; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Female; GTP-Binding Proteins; Mice; Molecular Sequence Data; Mutation; Ovarian Neoplasms; Pertussis Toxin; Phenotype; Phospholipases A; Phospholipases A2; Proto-Oncogene Proteins; Rats; Sequence Homology, Amino Acid; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Virulence Factors, Bordetella

The association of the production of CA125 with the cell cycle was investigated in two cell lines derived from human ovarian cancer, one from a serous cystadenocarcinoma (HTOA) and the other from a mucinous cystadenocarcinoma (RMUG-s). HTOA and RMUG-s cells secreted CA125 at about 50 and 30U/ml/10(5) cell/24hr, respectively, in the logarithmic growth phase and at about 75 and 100U/ml/10(5) cell/24hr in the steady phase. Analysis by FCM revealed that cultures of both cell lines cultured for 7 days contained more cells in the G0/G1 phase and less cells in the S phase than those cultured for 3 days. The positive rate of immunologically stained DNA polymerase alpha was 31% in HTOA cells and 39% in RMUG-s cells after cultivation of the cells for 3 days. The addition of EGF at 0.01, 0.1 or 1.0nM did not affect the production of CA125 in HTOA or RMUG-s cells while the addition of NaBT at 1, 3 and 5mM raised production in both cell lines as the dose rose. With RMUG-s cells, the addition of EGF at 0.01nM to the culture media accelerated both logarithmic and steady phase growth without a significant change in the production of CA125. In contrast, the addition of NaBT at 1mM suppressed growth, but tended to increase the production of CA125 per cell. With the effect of EGF on the cell cycle of both cell lines, cells in the S phase increased by about 20% as compared with the control, 48 hours after its addition at 0.01nM. In contrast, after cultivation for 48 hours in the presence of 1mM NaBT, cells in the S phase were decreased while those in the G0/G1 phase increased. The results presented above suggested the possibility that some factors other than the cell cycle were involved in the production of CA125. There also is close correlation between cells in the G0/G1 phase and the production of CA125 in the culture of human ovarian cancer cells.

Topics: Antigens, Tumor-Associated, Carbohydrate; Butyrates; Butyric Acid; Cell Cycle; Cystadenocarcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Tumor Cells, Cultured

The role of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) in the growth modulation of three human ovarian adenocarcinoma cell lines, PEO1, PEO4 and PEO14, has been examined by measuring responses of the cells growing in monolayer culture to exogenous addition of the growth factors. The presence of EGF receptors in the cell lines has been confirmed by ligand binding and immunocytochemical staining using a monoclonal antibody directed against the EGF receptor. The growth of all three cell lines was stimulated by both EGF and TGF-alpha. Dose-response effects were noted with the greatest growth stimulation occurring at concentrations between 0.1 and 10 nmol/l. The stimulatory effects of EGF and TGF-alpha were accompanied by changes in the cell cycle distribution as detected by flow cytometric analysis. It is concluded that EGF and TGF-alpha are important growth regulators in these EGF-receptor positive ovarian cancer cells.

Topics: Adenocarcinoma; Cell Cycle; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Neoplasm Proteins; Ovarian Neoplasms; Transforming Growth Factor alpha

To identify polypeptide growth factors for human teratocarcinoma cells, we studied the malignant ovarian teratoma-derived cell line, PA-1, that grew autonomously in serum-free medium. Medium conditioned by undifferentiated PA-1 cells strongly stimulated proliferation of the mouse mammary tumor cell line, GR 2H6, which is responsive to epidermal growth factor (EGF) and insulinlike growth factor-I (IGF-I). After ammonium sulfate precipitation, PA-1 conditioned medium was analyzed by anion exchange chromatography and bioassay of elution fractions on GR 2H6 cells that were grown in medium deficient in either EGF or insulin. The results demonstrated that PA-1 CM contained factors that can substitute for EGF and IGF-I in stimulating growth of GR 2H6 cells. Western blots of peak mitogenic fractions revealed low molecular weight polypeptides that were immunoreactive with either anti-EGF or anti-IGF-I antibodies. Indirect immunofluorescence staining of PA-1 cells with monoclonal antibodies localized receptors for each growth factor, and binding of human EGF and IGF-I to these cells was quantified by radioreceptor assays. Secretion of factors closely related to EGF and IGF-I by PA-1 cells under serum-free conditions may provide a novel model system to study molecular mechanisms of autocrine growth stimulation in teratocarcinomas.

Topics: Animals; Antibodies; Blotting, Western; Child; Chromatography, Ion Exchange; Culture Media; Epidermal Growth Factor; ErbB Receptors; Female; Fluorescent Antibody Technique; Humans; Insulin-Like Growth Factor I; Mammary Neoplasms, Experimental; Mice; Ovarian Neoplasms; Peptides; Receptors, Cell Surface; Receptors, Somatomedin; Teratoma; Tumor Cells, Cultured

We examined 35 primary human ovarian adenocarcinomas for the presence of epidermal growth factor (EGF) receptor (EGFR) in plasma membranes from cancer tissues by using 125I-EGF as a ligand. Specific 125I-EGF bindings were observed in 20 (57%) of these 35 cases. Scatchard analysis showed a class of high affinity EGF receptor: Kd 5.0 +/- 1.0 x 10(-10) M; Bmax, 83.3 +/- 12.1 fmol/mg protein (mean +/- SE, n = 20). Northern analysis in polyadenylated RNA from 15 EGFR(+) cancers using pretransforming growth factor alpha (pre-TGF alpha), prepro-EGF complementary DNA, and pE7, a complementary DNA clone of human EGFR, as probes revealed that pre-TGF alpha and EGFR mRNAs but not prepro-EGF mRNA were expressed in all cases examined. Immunocytochemical studies using monoclonal antibodies (mAbs) against TGF alpha, EGF, and EGFR showed that TGF alpha and EGFR but not EGF proteins were present on ovarian cancer cells in all cases. These data suggested a possible TGF alpha/EGFR autocrine mechanism in EGFR(+) ovarian cancers. We, therefore, examined the biological significance of this autocrine mechanism by using primary monolayer cell cultures. In primary cultures from EGFR (+) cancers, TGF alpha added to the culture medium stimulated the [3H]thymidine incorporation dose dependently. Moreover, the addition of mAbs against TGF alpha and EGFR but not EGF inhibited [3H]thymidine incorporation dose dependently in EGFR(+) cancer cells. On the other hand, in primary cultures from EGFR(-) cancers, TGF alpha and anti-TGF alpha, -EGFR, and -EGF mAbs did not show any effects on [3H]thymidine incorporation. All these results suggested the possible crucial role of a TGF alpha/EGFR autocrine growth mechanism in primary human ovarian cancers which express EGFR.

Topics: Adenocarcinoma; Blotting, Northern; DNA; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Humans; Ovarian Neoplasms; RNA; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

Although transforming growth factor (TGF) alpha and epidermal growth factor (EGF) receptor (EGFR) autocrine mechanism is widely demonstrated in many kinds of cancers, its biological significances still remain circumstantial. We critically assessed the significance of this mechanism on the growth of an ovarian cancer cell line. Northern blot analysis in polyadenylated RNA isolated from cells by using 32P-labeled pre-TGF alpha, EGRF, and prepro-EGF complementary DNAs as probes revealed that pre-TGF alpha and EGFR but not prepro-EGF gene transcripts were expressed in the cell. TGF alpha and EGFR but not EGF proteins were observed by immunocytochemical stainings, using monoclonal antibodies against human TGF alpha, EGFR, and EGF, respectively. This cell line possessed a class of high affinity EGF receptor by 125I-EGF binding studies; Kd being 2.9 x 10(-10) M and Bmax to be 7.7 x 10(4) sites/cell. As much as 1.12 +/- 0.14 ng (SD; n = 3)/10(7) cells/24 h of TGF alpha was secreted in the conditioned media. These results suggested the expression of a TGF alpha/EGFR autocrine mechanism in this cell line. We, therefore, assessed the biological significance of this mechanism on the growth of this cell line in serum-free monolayer cell cultures. Although 0.1, 1.0, and 10 nM concentrations of TGF alpha did not show significant growth promotion, monoclonal antibodies against TGF alpha and EGFR but not EGF significantly inhibited cell growth. All these data suggested the biological importance of a TGF alpha/EGFR autocrine mechanism on the growth of this cell line in vitro.

Topics: Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Blotting, Northern; Cell Division; Cell Line; Culture Media, Serum-Free; Cystadenoma; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Ovarian Neoplasms; Poly A; Protein Precursors; RNA; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta

Topics: Cisplatin; Colforsin; Drug Resistance; Drug Synergism; Epidermal Growth Factor; Female; Humans; Ovarian Neoplasms; Protein Kinases; Signal Transduction

Epidermal growth factor and transforming growth factor alpha are two peptides which bind to the epidermal growth factor receptor. One hundred and seventy-four samples from 133 patients with ovarian cancer were examined for EGF and TGF alpha. EGF was detected in only 27.6% of samples while TGF alpha was present in 88.5%. The median values for TGF alpha presence were at least 10-fold greater than those of EGF. There was no statistical difference between either TGF alpha or EGF levels and degree of differentiation of the tumours. There was no statistical difference between stage three and four in relation to concentration of either peptide. Median concentration did not differ significantly among the histological sub-groups.

Topics: Carcinoma; Cell Differentiation; Epidermal Growth Factor; Female; Humans; Middle Aged; Ovarian Neoplasms; Radioimmunoassay; Transforming Growth Factor alpha

We have elucidated the importance of a transforming growth factor (TGF) alpha and epidermal growth factor receptor autocrine mechanism on the growth of a human ovarian serous cystadenocarcinoma-derived cell line (SHIN-3) in vitro. In this study, we studied the biological significance of this autocrine mechanism in vivo using female athymic nude (nu/nu) mice. We measured the mouse plasma epidermal growth factor and TGF alpha levels by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Plasma epidermal growth factor concentrations were remarkably decreased by sialoadenectomy (Sx): 410 +/- 65 (SE) pg/ml (n = 10) in intact animals; and undetectable in Sx mice (n = 5). Plasma TGF alpha levels were 90 and 40 pg/ml in intact and in Sx animals, respectively. Ten million SHIN-3 cells inoculated into nu/nu mice formed tumors in 100% of mice, and tumors grew progressively. Implantabilities and tumor growth rates of inoculated cells were not affected by Sx and even by Sx and anti-mouse epidermal growth factor antibody treatment. However, anti-TGF alpha monoclonal antibody (mAb) administered to SHIN-3 cell-inoculated Sx animals drastically reduced the tumor growth. Although 10(7) SHIN-3 cells formed tumors in this group, tumor growth was significantly inhibited by 10 micrograms of anti-TGF alpha mAb given 3 times a week, and growth inhibitions were more by 20 micrograms of anti-TGF alpha mAb. Moreover, as aggressive tumor growth as that in Sx animals was resumed by the cessation of anti-TGF alpha mAb treatments. All these data suggested the biological importance of a TGF alpha/epidermal growth factor receptor autocrine mechanism on the growth of this cell line in vivo.

Topics: Animals; Antibodies, Monoclonal; Cell Division; Cell Line; Cystadenoma; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Ovarian Neoplasms; Radioimmunoassay; Transforming Growth Factor alpha; Transplantation, Heterologous

The growth promoting properties of ascitic fluids, cyst fluids and peritoneal fluids from patients with ovarian malignancy, benign ovarian tumours and non-tumour related gynaecological conditions have been investigated using an ovarian carcinoma cell line (OAW 42), mesothelial cells (58MC) and rat kidney cells (NRK-49F). Colony stimulating activity (CSA) for tumour cells and transforming activity (TA) for mesothelial cells were weakly correlated, but whereas elevated TA was tumour-associated, CSA was not. However, TA was not cancer-associated and, although the difference between the mean TA values of benign and malignant cyst fluids was of borderline significance, some benign cyst fluids from cystadenomas showed high TA values. Higher levels of TA in the cystadenomas showed a significant correlation with the menopausal status of the patient and higher levels of TA in the malignant cyst fluid/peritoneal fluid groups were associated with more advanced disease. Results indicated that some fluids contained TGF-beta-like activity, but there was no direct evidence for the presence of TGF-alpha/EGF-like activity in the fluids. Heparin inhibited clonogenic growth of tumour cells but not mesothelial cells. The reduced CSA which was observed after treatment of fluids with both heparin and thrombin implicated coagulation factors in the manifestation of CSA. It was concluded that CSA in the fluids was due, at least partly, to fibrin coagulation, and TA was due to unknown growth factor(s) which may include TGF-beta-like activity. The results are discussed in the context of the aetiology of ovarian carcinoma, and the possible clinical significance of TA.

Topics: Animals; Ascitic Fluid; Blood Coagulation Factors; Body Fluids; Cell Division; Cell Transformation, Neoplastic; Colony-Stimulating Factors; Cysts; Epidermal Growth Factor; Female; Growth Substances; Heparin; Humans; Mice; Ovarian Neoplasms; Thrombin; Transforming Growth Factor beta; Tumor Cells, Cultured

Previously we have shown that epidermal growth factor acts as a mitogen for some, but not all, ovarian cancer cells in culture. In this study we examined the effect of epidermal growth factor on proliferation of normal human ovarian epithelial cells in monolayer culture. We found that epidermal growth factor stimulated twofold to fourfold increases in proliferation in epithelial cells from each of five normal ovaries (p less than 0.01). In addition, Scatchard analysis of binding of epidermal growth factor tagged with iodine 125 indicated the presence of high-affinity receptors in all of the ovarian epithelial cells and ovarian cancer cell lines. The number and affinity of receptors was similar in the normal epithelium and cancer cell lines, and there was no relationship between epidermal growth factor receptor number and responsiveness to epidermal growth factor. We conclude that human ovarian epithelial cells normally express epidermal growth factor receptors and that epidermal growth factor acts as a mitogen for these cells. Although the mitogenic response to epidermal growth factor often is attenuated in ovarian cancer cell lines, loss of responsiveness to epidermal growth factor does not appear to be due to decreased receptor expression.

Topics: Cells, Cultured; Epidermal Growth Factor; Epithelium; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Ovary; Thymidine

Cisplatin (DDP) is the most effective drug for the treatment of human ovarian cancer, but the mechanisms that determine sensitivity to the cytotoxic action of DDP are not well understood. Treatment of two human ovarian carcinoma cell lines with epidermal growth factor (EGF) simultaneously increased sensitivity to DDP and caused a persistent change in morphology in the absence of any mitogenic effect. Sensitization to DDP was shown to be dependent on both EGF concentration and EGF receptor number in C127 mouse fibroblasts expressing the human EGF receptor after transfection with a pBPV plasmid construct containing the human EGF receptor gene under control of the transferrin receptor 3'-inducible regulator. Sensitization of human ovarian carcinoma cells to DDP was not blocked by inhibition of protein synthesis. EGF did not enhance sensitivity to DDP or alter morphology in DDP-resistant human ovarian carcinoma cells despite the presence of functional EGF receptors on these cells. These results showed that elements of the signal transduction pathway activated by EGF determined cellular sensitivity to DDP, and that a DDP-resistant phenotype is associated with a defect in this signal transduction pathway.

Topics: Animals; Cell Line; Cisplatin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Mice; Ovarian Neoplasms; Signal Transduction; Time Factors; Tumor Cells, Cultured

We studied the effect of epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, and transforming growth factor-beta on proliferation of four epithelial ovarian cancer cell lines (OVCA 420, OVCA 429, OVCA 432, and OVCA 433). Epidermal growth factor stimulated growth of OVCA 429 cells (P = .0001) and OVCA 433 cells (P = .0002). Platelet-derived growth factor did not stimulate growth of any of the cell lines. Fibroblast growth factor stimulated growth of OVCA 420 cells (P = .003). Transforming growth factor-beta inhibited growth of OVCA 420 cells (P = .0001), OVCA 432 cells (P = .003), and OVCA 433 cells (P = .004). To detect production of known growth factors by the cancer cell lines, we tested the effect of cancer cell-conditioned media on proliferation of cell lines known to respond to growth factors. Only media exposed to OVCA 433 cells were found to contain activity that mimicked one of the known growth factors (transforming growth factor-beta). These results suggest that individual ovarian cancers vary widely in their response to and production of known peptide growth factors. Finally, we found that OVCA 429-conditioned medium significantly inhibited proliferation of mitogen-stimulated lymphocytes (P less than .0001). The characteristics of this immunosuppressive factor were distinct from those of transforming growth factor-beta. Production of this factor by an immortalized cell line provides a unique opportunity to identify an immunosuppressive substance associated with ovarian cancer.

Topics: Cell Division; Culture Media; Epidermal Growth Factor; Female; Fibroblast Growth Factors; Growth Substances; Humans; Ovarian Neoplasms; Platelet-Derived Growth Factor; Pregnancy; Transforming Growth Factors; Tumor Cells, Cultured

A cDNA encoding transforming growth factor type alpha (TGF alpha) was fused to the 5' end of a gene encoding a modified form of Pseudomonas exotoxin A (PE), which is devoid of the cell recognition domain (domain Ia). The chimeric molecule, termed TGF alpha-PE40, was expressed in Escherichia coli and isolated from the periplasm or inclusion bodies depending on the construction expressed. TGF alpha-PE40 was found to be extremely cytotoxic to cells displaying epidermal growth factor (EGF) receptors. Comparison with a similar molecule in which TGF alpha was placed at the carboxyl end of PE40 demonstrated the importance of the position of the cell recognition element; TGF alpha-PE40 was found to be about 30-fold more cytotoxic to cells bearing EGF receptors than PE40-TGF alpha. In addition, TGF alpha-PE40 was shown to be extremely cytotoxic to a variety of cancer cell lines including liver, ovarian, and colon cancer cell lines, indicating high levels of EGF receptor expression in these cells.

Topics: ADP Ribose Transferases; Bacterial Toxins; Base Sequence; Binding, Competitive; Carcinoma, Hepatocellular; Cell Survival; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Escherichia coli; Exotoxins; Female; Gene Expression; Liver Neoplasms; Molecular Sequence Data; Ovarian Neoplasms; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Structure-Activity Relationship; Transforming Growth Factor alpha; Transforming Growth Factors; Tumor Cells, Cultured; Virulence Factors

In this study we investigated the presence of epidermal growth factor receptors (EGF-R) and the tissue levels of EGF-like factors (EGF-F) in ovarian, endometrial, cervical and breast carcinomas. EGF-R were found in 33/40 (83%) cervical, 15/26 (58%) endometrial, 64/141 (45%) ovarian, and 19/59 (33%) breast carcinomas. The highest number of EGF-R binding sites was detected in cervical carcinomas followed by endometrial, breast and ovarian carcinomas. The tissue concentrations of EGF-like factors, were investigated in extracts of 63 ovarian, 25 breast, 12 cervical, 14 endometrial carcinomas and in 21 biopsies of nonmalignant tissue such as myometrium and ovaries. The extracts of nonmalignant tissues had a mean EGF-F level of 1.5 +/- 0.7 ng/mg with a concentration range from 0 to 4 ng/mg. The mean EGF-F levels of malignant tissues were: ovarian carcinomas 4.2 +/- 1.5 ng/mg (range 0-15 ng), endometrial carcinomas 4.5 +/- 1.7 ng/mg (range 0-12 ng), cervical carcinomas 4.15 +/- 1.1 ng/mg (range 0-8) and breast carcinomas 3.16 +/- 1.1 ng/mg (range 0-10 ng). About 30% ovarian, endometrial and cervical carcinomas and 16% breast carcinomas, respectively, had enhanced EGF levels from 5 ng/mg to 15 ng/mg compared to nonmalignant tissues. The EGF-F of tissue extracts consists of EGF and transforming growth factor TGF alpha) as shown by the results of EGF and TGF alpha radioimmunoassays. It is assumed that in some tumors the EGF-F tissue levels influence the number of biochemically detectable EGF binding sites.

Topics: Binding Sites; Breast Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Prognosis; Radioimmunoassay; Transforming Growth Factors; Uterine Cervical Neoplasms; Uterine Neoplasms

Results are presented which show that mesothelial cells (MC) from ovarian cancer patients can both stimulate and inhibit the clonogenic growth of ovarian tumour cells (TC) in a dose-dependent fashion. TC lines from both non-ovarian and ovarian tumours were variable in their response to MC. Colony formation was rarely induced when the TC population was non-clonogenic and a bladder cell line showed inhibition of colony formation in the presence of MC. Primary tumour cultures from ovarian cancer patients also showed a variable response to MC. Fibroblasts from malignant, benign and non-neoplastic sources were significantly less effective in stimulating the clonogenic growth of responsive cell lines. Conditioned medium was a poor substitute for the presence of intact viable cells, and distance between feeder cell and TC was an important factor in determining the magnitude of response. A significant relationship between the feeder effect of MC and their proliferation in soft agar was observed when epidermal growth factor was used in the medium. The relevance of the findings in the context of the pattern of spread of ovarian cancer is discussed.

Topics: Ascites; Cell Communication; Cell Division; Epidermal Growth Factor; Epithelium; Female; Humans; Mesoderm; Ovarian Neoplasms; Tumor Cells, Cultured

We have studied the concentration of epidermal growth factor receptors (EGF-R) in 115 different malignant ovarian tumors (101 ovarian carcinomas) and EGF-like factors (EGF-F) in tissue extracts of 63 different ovarian carcinomas and 20 non-malignant tissues. 36% of ovarian carcinomas are EGF-R positive. The calculated mean EGF-F level of 4.2 +/- 1.5 ng mg-1 (range: 0-15 ng mg-1) in ovarian carcinomas is significantly enhanced compared to 1.5 +/- 0.7 ng mg-1 (range: 0-4 ng mg-1) of non-malignant tissue extracts. The correlation between EGF-R positive as well as negative ovarian carcinomas and the results of a primary chemotherapy of advanced ovarian carcinomas (n = 92) revealed a significantly higher remission rate of EGF-R positive tumors (66%) compared to EGF-R negative cases (23%). 84% of tumors with progressive disease were EGF-R negative. The mean EGF-F levels were calculated for prognostic subgroups of ovarian carcinomas. Increased EGF-F levels are significantly associated with progressive disease compared to all patients or the remission group. 15/16 cases with EGF-F levels greater than 5 ng mg-1 showed progressive disease. The overall survival time of patients with tumor tissue EGF-F levels greater than 3.5 ng mg-1 was worse than that of patients with low EGF-F levels. Multivariate analysis showed that the EGF-F level was, after grading, the second most important factor for predicting overall survival.

Topics: Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Placenta; Pregnancy; Prognosis

Epidermal growth factor (EGF) receptor binding properties were examined in spontaneous ovarian granulosa cell (GC) tumors from SWR and SWR-derived strains of mice. EGF binding was measured at room temperature in tissue homogenates from GC tumors and normal ovaries from adult randomly cycling mice. GC tumor tissue displayed significantly increased EGF binding and 2 receptor populations (R1 and R2). Normal ovarian tissue appeared to have only one receptor population with a dissociation constant (KD) similar to the R1 (high-affinity) receptor in GC tumors. In subsequent experiments, GC tumor and normal granulosa cells from immature mice were analyzed in primary cultures for EGF binding, immunofluorescence microscopy for receptors, and cell proliferation. After 24 hr in culture, the GC tumors bound 10-fold more EGF/micrograms protein than did normal granulosa cells. GC tumor cells, but not normal granulosa cells, showed specific immunofluorescence when reacted with a polyclonal antibody to mouse EGFR. During 96 hr in culture, GC tumor cells, but not normal cells, showed a significant proliferative response to EGF. In conclusion, the EGF binding capacity is markedly increased in GC tumor cells and the proliferation data suggest that this growth factor supports tumor growth in the SWR model system.

Topics: Animals; Cell Division; Epidermal Growth Factor; ErbB Receptors; Female; Granulosa Cell Tumor; Mice; Ovarian Neoplasms; RNA, Messenger

Direct inhibitory effects of calmodulin inhibitors on human ovarian cancer cell proliferation in vitro were examined. All calmodulin inhibitors used in this study inhibited proliferation of HR cells derived from human serous cystadenocarcinoma of the ovary in a dose-dependent manner. The degree of inhibition by melittin and W-5 was most marked and was followed by that of W-7, R24571, and bepridil. HR cell proliferation was dose-dependently stimulated by epidermal growth factor (EGF). The stimulatory effects seemed to be a result of the binding of EGF to HR cells. Binding of EGF was significantly inhibited by W-5, R24571, and melittin. These results suggest that inhibition by calmodulin inhibitors of cancer cell proliferation may result in part from the interference in the binding of EGF to the cells.

Topics: Biomechanical Phenomena; Calmodulin; Cell Division; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Humans; Ovarian Neoplasms; Tumor Cells, Cultured

Glycosphingolipids added exogenously to 3T3 cells in culture were shown to inhibit cell growth, alter the membrane affinity to platelet-derived growth factor binding, and reduce platelet-derived growth factor-stimulated membrane phosphorylation (Bremer, E., Hakomori, S., Bowen-Pope, D. F., Raines, E., and Ross, R. (1984) J. Biol. Chem. 259, 6818-6825). This approach has been extended to the epidermal growth factor (EGF) receptor of human epidermoid carcinoma cell lines KB and A431. GM3 and GM1 gangliosides inhibited both KB cell and A431 cell growth, although GM3 was a much stronger inhibitor of both KB and A431 cell growth. Neither GM3 nor GM1 had any affect on the binding of 125I-EGF to its cell surface receptor. However, GM3 and, to a much lower extent, GM1 were capable of inhibiting EGF-stimulated phosphorylation of the EGF receptor in membrane preparations of both KB and A431 cells. Further characterization of GM3-sensitive receptor phosphorylation was performed in A431 cells, which had a higher content of the EGF receptor. The following results were of particular interest. (i) EGF-dependent tyrosine phosphorylation of the EGF receptor and its inhibition by GM3 were also demonstrated on isolated EGF receptor after adsorption on the anti-receptor antibody-Sepharose complex, and the receptor phosphorylation was enhanced on addition of phosphatidylethanolamine. (ii) Phosphoamino acid analysis of the EGF receptor indicated that the reduction of phosphorylation induced by GM3 was entirely in the phosphotyrosine and not in the phosphoserine nor phosphothreonine content. (iii) The inhibitory effect of GM3 on EGF-dependent receptor phosphorylation could be reproduced in membranes isolated from A431 cells that had been cultured in medium containing 50 nmol/ml GM3 to effect cell growth inhibition. The membrane fraction isolated from such growth-arrested cells was found to be less responsive to EGF-stimulated receptor phosphorylation. These results suggest that membrane lipids, especially GM3, can modulate EGF receptor phosphorylation in vitro as well as in situ.

Topics: Amino Acids; Animals; Carcinoma, Squamous Cell; Cattle; Cell Division; Cell Line; Depression, Chemical; Dogs; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Humans; Immunosorbent Techniques; Mice; Mouth Neoplasms; Ovarian Neoplasms; Phosphorylation; Phosphotyrosine; Receptors, Cell Surface; Tyrosine

The mechanisms of ectopic enzyme and enzyme inhibitor-production in neoplastic tissues were investigated. No evidence was obtained to suggest any difference in the structural genes of amylase-producing tumors and normal lymphocytes. mRNA sequence coding for amylase precursor in tumor tissues was identical to that of salivary amylase, suggesting that the amylase in amylase-producing tumors was identical to, or closely resembled the amylase in salivary gland. High incidence of elevation of serum pancreatic secretory trypsin inhibitor (PSTI) was observed in patients with various malignant tumors. PSTI-positive malignant cells were also frequently found in malignant tissues. A comparison of human PSTI mRNA sequence with mouse epidermal growth factor (EGF) mRNA sequence showed that they were 46% homologous. Human PSTI stimulated [3H]thymidine incorporation into DNA in human fibroblasts at concentrations present in human serum.

Topics: Amylases; Animals; Base Sequence; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Mice; Mice, Nude; Neoplasms; Ovarian Neoplasms; RNA, Messenger; Salivary Glands; Thymoma; Thymus Neoplasms; Trypsin Inhibitor, Kazal Pancreatic; Trypsin Inhibitors

We describe the properties of two monoclonal antibodies produced to a synthetic peptide consisting of residues 985 to 996 from the cytoplasmic domain of the epidermal growth factor (EGF) receptor. We have examined a group of ten human tumors including cervical, ovarian, and vulval carcinomas for expression of EGF receptors by immunohistological staining using one of these antibodies and another monoclonal antibody to the extracellular domain of the molecule. The tumors were examined using a sensitive amplified enzyme system and a less sensitive indirect staining method. There was generally a good correlation in staining intensity with the two monoclonal antibody reagents. Both antibodies showed strong staining of squamous cell carcinomas and usually weak or heterogeneous patterns with the adenocarcinomas. Samples of each tumor were solubilized in detergent and analyzed for the presence of functional EGF receptors by immunoprecipitation and autophosphorylation. Three of the squamous cell tumors gave labeled bands, Mr 170,000, on sodium dodecyl sulfate:polyacrylamide gels. DNA was extracted from seven of the tumors and digested with two restriction endonucleases, and the fragments were analyzed on Southern blots using probes representing the extracellular and cytoplasmic domains of the molecule. The tumor DNA showed no apparent rearrangements or amplifications when compared to the EGF receptor gene in human placental DNA. These results suggest that there is a high level of EGF receptors on some squamous cell tumors.

Topics: Antibodies, Monoclonal; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation; Humans; Ovarian Neoplasms; Phosphoproteins; Phosphorylation; Receptors, Cell Surface; Uterine Cervical Neoplasms; Vulvar Neoplasms

Epidermal growth factor (EGF)-like factors with EGF competing and cell growth stimulating activity were investigated in malignant and nonmalignant tissues. About 37% of ovarian carcinomas present an increased factor activity between 9.0 and 19.3 ng EGF units/mg protein. In one tumor 175.0 ng EGF units/mg protein were found. In extracts of nonmalignant tissues, the factor concentration was about 1.0-6.4 ng EGF units/mg protein. Isoelectric focusing was performed to characterize these factors. In normal ovaries and ovarian carcinomas, factors with EGF competing activity focus at pH 8.0-9.0, pH 5.7-6.3, and pH 3.6-4.9. In ovarian carcinomas, an additional peak with EGF competing and cell growth stimulating activity was found between pH 6.5 and 7.2. Similar results could be achieved in other malignant tissues investigated. These data indicate the presence of EGF-like factors. EGF itself focuses at pH 4.6 (G. Carpenter and S. Cohen, Annu. Rev. Biochem., 48: 193-216, 1979). Specific EGF binding was determined in 12 ovarian carcinomas. In five of these cases EGF receptors could be detected. In the EGF receptor positive carcinomas, the content of EGF-like growth factors varied between 0 and 9 ng EGF units/mg protein. In EGF receptor negative cases, the content of EGF-like growth factors varied between 0 and 19.3 ng EGF units/mg protein. The clinical data of 19 patients are also demonstrated.

Topics: Animals; Binding, Competitive; Biological Assay; Breast Neoplasms; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Growth Substances; Humans; Isoelectric Point; Melanoma; Mice; Ovarian Neoplasms; Radioligand Assay; Receptors, Cell Surface; Sarcoma

We investigated the ability of the teratocarcinoma-derived, epithelial-type cell line 1H5 to differentiate into either of the two pathways to primary endoderm, and tested the hypothesis that 1H5 represents a state similar to primitive endoderm in the late 4th-day blastocyst. Like other endodermal cell types, 1H5 cells mixed with embryonal-carcinoma cells sort out into "embryoid bodies" or structures that resemble 4th-day mouse embryos. The epithelial line conforms morphologically and biochemically to the few known characteristics typical of primitive endoderm. The present study demonstrates that the formation in vitro of overt visceral endoderm is readily achieved. The spontaneous arrangement of the cells into a cystic form is followed by the appearance of several markers of visceral endoderm, most notably alphafetoprotein, which is detected when 1H5 cells are cultured either in the presence of retinoic acid or when the cells interact with embryonal-carcinoma cells in a specific spatial arrangement after sorting out. However, some less specific properties of visceral endoderm are not expressed. Although 1H5 differentiates histologically into parietal-like endoderm in the tumor form, parietal cells cannot yet be identified with certainty in vitro because of the paucity of parietal-specific markers. The 1H5 cell line could provide a useful system for studying the characteristics and mechanisms underlying visceral-endoderm differentiation in vitro, since it has the distinct advantage that homogeneous cultures are produced, in contrast to other teratocarcinoma cell lines such as F9 which differentiate into a mixture of cell types.

Topics: Animals; Cell Aggregation; Cell Differentiation; Cell Line; Clone Cells; Culture Media; Endoderm; Epidermal Growth Factor; ErbB Receptors; Female; Mice; Ovarian Neoplasms; Plasminogen Activators; Receptors, Cell Surface; Teratoma

The factors controlling the growth of human tumor cells in soft agar are poorly understood. However, it has been demonstrated that serum provides factors which promote anchorage-independent growth. We tested 58 tumor specimens, which were obtained from patients with adenocarcinoma of the lung, colon, ovary or squamous cell carcinoma, for their ability to form colonies in soft agar in serum-free or serum-supplemented media. The cells were unable to replicate, and none of the hormones or growth factors tested: insulin (I), transferrin (T), selenium (S), estradiol (E), hydrocortisone (H) or epidermal growth factor (EGF) could substitute for serum. Examination of the serum dose-response curves indicated that growth factors reduced the serum concentrations needed to support anchorage-independent growth. The addition of the supplements and the lowering of serum concentrations increased cloning efficiencies (C.E.) in 38/51 trials, when cells were able to grow initially. The addition of ITS increased C.E. in 18/21 cases, HITES in 15/17 cases and EGF in 12/18 cases as compared to controls. ITS and HITES increased the number of colonies only when serum was the limiting factor. EGF, however, increased the number of colonies even when serum was not the limiting factor. The ability of the supplements to enhance growth could not be correlated to tumor type or initial cloning efficiencies. However, in only 1/25 cases were cells that were unable to form colonies under standard conditions induced to form colonies in the presence of the growth factors. Normal and tumor-derived human fibroblasts did not form colonies in soft agar in the presence of these growth factors. The results suggest that human tumor cells may require the presence of serum-derived factors for growth in soft agar.

Topics: Adenocarcinoma; Agar; Blood; Carcinoma, Squamous Cell; Cell Division; Clone Cells; Colonic Neoplasms; Epidermal Growth Factor; Estradiol; Female; Humans; Hydrocortisone; Insulin; Lung Neoplasms; Ovarian Neoplasms; Selenium; Transferrin

Topics: Animals; Cell Division; Cell Line; Cell Membrane; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Neoplasm Metastasis; Neoplasms, Experimental; Ovarian Neoplasms; Peptides; Rats; Transferrin