sphingosine-1-phosphate and Ovarian-Neoplasms

Ovarian cancer is the most lethal gynaecological malignancy. It is commonly diagnosed at advanced stage when it has metastasised to the abdominal cavity and treatment becomes very challenging. While current standard therapy involving debulking surgery and platinum + taxane-based chemotherapy is associated with high response rates initially, the large majority of patients relapse and ultimately succumb to chemotherapy-resistant disease. In order to improve survival novel strategies for early detection and therapeutics against treatment-refractory disease are urgently needed. A promising new target against ovarian cancer is the sphingolipid pathway which is commonly hijacked in cancer to support cell proliferation and survival and has been shown to promote chemoresistance and metastasis in a wide range of malignant neoplasms. In particular, the sphingosine kinase 1-sphingosine 1-phosphate receptor 1 axis has been shown to be altered in ovarian cancer in multiple ways and therefore represents an attractive therapeutic target. Here we review the roles of sphingolipids in ovarian cancer progression, metastasis and chemoresistance, highlighting novel strategies to target this pathway that represent potential avenues to improve patient survival.

Topics: Animals; Female; Humans; Lysophospholipids; Neoplasm Proteins; Ovarian Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors

Sphingosine-1-phoshate (S1P) is a membrane sphingolipid involved in several physiological processes, including cell proliferation, tissue growth, cell survival and migration, inflammation, vasculogenesis, and angiogenesis. Herein, we review the most critical effects of S1P on ovarian function, including its physiological and pathophysiological effects. Based on the available evidence, S1P plays an important role in ovarian physiology, participating as an essential stimulator of follicular development in both the preantral and antral phases, as well as in ovulation and corpus luteum development. Moreover, S1P may be a good cytoprotective agent against cancer treatment side-effects (chemotherapy with or without radiation therapy). In the future, this compound may be given for fertility preservation to women undergoing cancer treatment. However, further studies are required to confirm its efficacy in ovarian protection and also its safety in terms of cancer prognosis, given the biological action of the compound. Under- or over-production of S1P may be related to ovarian pathologies.

Topics: Animals; Cell Proliferation; Corpus Luteum; Female; Fertility Preservation; Humans; Lysophospholipids; Ovarian Diseases; Ovarian Follicle; Ovarian Neoplasms; Ovary; Sphingosine; Sphingosine-1-Phosphate Receptors

Sphingosine 1-phosphate (S1P) is an important signaling regulator involved in tumor progression in multiple neoplasms. However, the role of S1P in the pathogenesis of ovarian cancer remains unclear. Herein, we summarize recent advances in understanding the impact of S1P signaling in ovarian cancer progression. S1P, aberrantly produced in ovarian cancer patients, is involved in the regulation of key cellular processes that contribute to ovarian cancer initiation and progression. Moreover, agents that block the S1P signaling pathway inhibit ovarian cancer cell growth or induce apoptosis. Hence, current evidence suggests that S1P may become a potential molecular target for ovarian cancer therapy.

Topics: Animals; Antineoplastic Agents; Female; Humans; Lysophospholipids; Molecular Targeted Therapy; Ovarian Neoplasms; Signal Transduction; Sphingosine

Topics: Animals; Antineoplastic Agents; Cell Hypoxia; Cyclooxygenase 2; Diterpenes; Female; Humans; Interleukins; Intracellular Signaling Peptides and Proteins; Lysophospholipids; Membrane Lipids; Mice; Multienzyme Complexes; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neovascularization, Pathologic; Ovarian Neoplasms; Peptide Hydrolases; Phosphodiesterase I; Phospholipases A; Phosphoric Diester Hydrolases; Pyrophosphatases; Receptors, Lysophosphatidic Acid; Signal Transduction; Sphingosine; Vascular Endothelial Growth Factor A

Topics: Antineoplastic Agents; Cell Hypoxia; Drug Delivery Systems; Female; Forecasting; Humans; Lysophospholipids; Multienzyme Complexes; Neoplasm Proteins; Ovarian Neoplasms; Phosphodiesterase I; Phospholipases A2; Phospholipases A2, Cytosolic; Phosphoric Diester Hydrolases; Pyrophosphatases; Receptors, Lysophosphatidic Acid; Signal Transduction; Sphingosine

The role of phospholipid signaling in ovarian cancer is poorly understood. Sphingosine-1-phosphate (S1P) is a bioactive metabolite of sphingosine that has been associated with tumor progression through enhanced cell proliferation and motility. Similarly, sphingosine kinases (SPHK), which catalyze the formation of S1P and thus regulate the sphingolipid rheostat, have been reported to promote tumor growth in a variety of cancers. The findings reported here show that exogenous S1P or overexpression of SPHK1 increased proliferation, migration, invasion, and stem-like phenotypes in ovarian cancer cell lines. Likewise, overexpression of SPHK1 markedly enhanced tumor growth in a xenograft model of ovarian cancer, which was associated with elevation of key markers of proliferation and stemness. The diabetes drug, metformin, has been shown to have anticancer effects. Here, we found that ovarian cancer patients taking metformin had significantly reduced serum S1P levels, a finding that was recapitulated when ovarian cancer cells were treated with metformin and analyzed by lipidomics. These findings suggested that in cancer the sphingolipid rheostat may be a novel metabolic target of metformin. In support of this, metformin blocked hypoxia-induced SPHK1, which was associated with inhibited nuclear translocation and transcriptional activity of hypoxia-inducible factors (HIF1α and HIF2α). Further, ovarian cancer cells with high SPHK1 were found to be highly sensitive to the cytotoxic effects of metformin, whereas ovarian cancer cells with low SPHK1 were resistant. Together, the findings reported here show that hypoxia-induced SPHK1 expression and downstream S1P signaling promote ovarian cancer progression and that tumors with high expression of SPHK1 or S1P levels might have increased sensitivity to the cytotoxic effects of metformin. IMPLICATIONS: Metformin targets sphingolipid metabolism through inhibiting SPHK1, thereby impeding ovarian cancer cell migration, proliferation, and self-renewal.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Movement; Female; Humans; Hypoglycemic Agents; Hypoxia-Inducible Factor 1, alpha Subunit; Lysophospholipids; Metformin; Mice; Mice, Nude; Molecular Targeted Therapy; Ovarian Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Xenograft Model Antitumor Assays

Epithelial ovarian carcinomas account for more than 90% of human ovarian cancers and have become the primary cause of death for gynecological malignancies. Unlimited cell proliferation and resistance to cell apoptosis contribute to the development of ovarian cancers. However, the underlying mechanisms involved in these processes in epithelial ovarian carcinomas are yet poorly understood. In the present study, we examined the Hippo signaling gene expression and investigated the effects of Sphingosine 1-phosphate (S1P) on cell proliferation and the underlying mechanisms in human ovarian cancer cell lines, OVCAR3 and SKOV3. Our results demonstrate that S1P disrupts Hippo signaling by reducing YAP phosphorylation and increasing the expression of CCN1 and CCN2 in both ovarian cancer cells. Furthermore, the increase in CCN1/CCN2 expression contributes to the S1P-induced increase in cancer cell proliferation.

Topics: Active Transport, Cell Nucleus; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Connective Tissue Growth Factor; Cysteine-Rich Protein 61; Female; Gene Expression Regulation, Neoplastic; Hippo Signaling Pathway; Humans; Lysophospholipids; Nuclear Proteins; Ovarian Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Protein Serine-Threonine Kinases; Signal Transduction; Sphingosine; Transcription Factors

Sphingosine kinase 1 (SPHK1), the enzyme that produces sphingosine 1 phosphate (S1P), is known to be highly expressed in many cancers. However, the role of SPHK1 in cells of the tumor stroma remains unclear. Here, we show that SPHK1 is highly expressed in the tumor stroma of high-grade serous ovarian cancer (HGSC), and is required for the differentiation and tumor promoting function of cancer-associated fibroblasts (CAFs). Knockout or pharmacological inhibition of SPHK1 in ovarian fibroblasts attenuated TGF-β-induced expression of CAF markers, and reduced their ability to promote ovarian cancer cell migration and invasion in a coculture system. Mechanistically, we determined that SPHK1 mediates TGF-β signaling via the transactivation of S1P receptors (S1PR2 and S1PR3), leading to p38 MAPK phosphorylation. The importance of stromal SPHK1 in tumorigenesis was confirmed in vivo, by demonstrating a significant reduction of tumor growth and metastasis in SPHK1 knockout mice. Collectively, these findings demonstrate the potential of SPHK1 inhibition as a novel stroma-targeted therapy in HGSC.

Topics: Animals; Apoptosis; Blotting, Western; Cell Differentiation; Cell Movement; Cell Proliferation; Cystadenocarcinoma, Serous; Female; Fibroblasts; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Lysophospholipids; Mice; Mice, Knockout; Myofibroblasts; Neoplasm Grading; Ovarian Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prognosis; Real-Time Polymerase Chain Reaction; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sphingosine; Survival Rate; Transforming Growth Factor beta; Tumor Cells, Cultured

Currently, conventional cancer treatment regimens often rely upon highly toxic chemotherapeutics or target oncogenes that are variably expressed within the heterogeneous cell population of tumors. These challenges highlight the need for novel treatment strategies that (1) are non-toxic yet able to at least partially reverse the aggressive phenotype of the disease to a benign or very slow-growing state, and (2) act on the cells independently of variably expressed biomarkers. Using a label-independent rapid microfluidic cell manipulation strategy known as contactless dielectrophoresis (cDEP), we investigated the effect of non-toxic concentrations of two bioactive sphingolipid metabolites, sphingosine (So), with potential anti-tumor properties, and sphingosine-1-phosphate (S1P), a tumor-promoting metabolite, on the intrinsic electrical properties of early and late stages of mouse ovarian surface epithelial (MOSE) cancer cells. Previously, we demonstrated that electrical properties change as cells progress from a benign early stage to late malignant stages. Here, we demonstrate an association between So treatment and a shift in the bioelectrical characteristics of late stage MOSE (MOSE-L) cells towards a profile similar to that of benign MOSE-E cells. Particularly, the specific membrane capacitance of MOSE-L cells shifted toward that of MOSE-E cells, decreasing from 23.94 ± 2.75 to 16.46 ± 0.62 mF m(-2) after So treatment, associated with a decrease in membrane protrusions. In contrast, S1P did not reverse the electrical properties of MOSE-L cells. This work is the first to indicate that treatment with non-toxic doses of So correlates with changes in the electrical properties and surface roughness of cells. It also demonstrates the potential of cDEP to be used as a new, rapid technique for drug efficacy studies, and for eventually designing more personalized treatment regimens.

Topics: Animals; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Disease Progression; Electrophoresis; Female; Lysophospholipids; Mice; Microfluidics; Models, Biological; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Phenotype; Sphingosine

Cancer progression is associated with an increased deformability of cancer cells and reduced resistance to mechanical forces, enabling motility and invasion. This is important for metastases survival and outgrowth and as such could be a target for chemopreventive strategies. In this study, we determined the differential effects of exogenous sphingolipid metabolites on the elastic modulus of mouse ovarian surface epithelial cells as they transition to cancer. Treatment with ceramide or sphingosine-1-phosphate in non-toxic concentrations decreased the average elastic modulus by 21% (p≤ 0.001) in transitional and 15% (p≤ 0.02) in aggressive stages while exerting no appreciable effect on non-malignant cells. In contrast, sphingosine treatment on average increased the elastic modulus by 33% (p≤ 0.0002) in aggressive cells while not affecting precursor cells. These results indicate that tumor-supporting sphingolipid metabolites act by making cells softer, while the anti-cancer metabolite sphingosine partially reverses the decreased elasticity associated with cancer progression. Thus, sphingosine may be a valid alternative to conventional chemotherapeutics in ovarian cancer prevention or treatment.

Topics: Animals; Biomechanical Phenomena; Cell Differentiation; Cell Line; Cell Line, Tumor; Ceramides; Disease Progression; Elastic Modulus; Female; Lysophospholipids; Mice; Microscopy, Atomic Force; Neoplasm Metastasis; Ovarian Neoplasms; Software; Sphingolipids; Sphingosine

Bioactive sphingolipid metabolites have emerged as important lipid second messengers in the regulation of cell growth, death, motility and many other events. These processes are important in cancer development and progression; thus, sphingolipid metabolites have been implicated in both cancer development and cancer prevention. Despite recent considerable progress in understanding the multi-faceted functions of these bioactive metabolites, little is known about their influence on the biomechanical property of cells. The biomechanical properties of cancer cells change during progression with aggressive and invasive cells being softer compared to their benign counterparts. In this paper, we investigated the effects of exogenous sphingolipid metabolites on the Young's modulus and cytoskeletal organization of cells representing aggressive ovarian cancer. Our findings demonstrate that the elasticity of aggressive ovarian cancer cells decreased ∼15% after treatment with ceramide and sphingosine-1-phosphate. In contrast, sphingosine treatment caused a ∼30% increase in the average elasticity which was associated with a more defined actin cytoskeleton organization. This indicates that sphingolipid metabolites differentially modulate the biomechanic properties of cancer cells which may have a critical impact on cancer cell survival and progression, and the use of sphingolipid metabolites as chemopreventive or chemo-therapeutic agents.

Topics: Animals; Biomechanical Phenomena; Cell Differentiation; Ceramides; Female; Humans; Lysophospholipids; Mice; Microscopy, Atomic Force; Models, Statistical; Ovarian Neoplasms; Sphingolipids; Sphingosine; Tumor Cells, Cultured

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.

Topics: Apoptosis; Autophagy; Caspases; Cell Line, Tumor; Cisplatin; Cytoprotection; Drug Screening Assays, Antitumor; Female; Fingolimod Hydrochloride; Humans; Inhibitory Concentration 50; Lysophospholipids; Necrosis; Ovarian Neoplasms; Propylene Glycols; Signal Transduction; Sphingosine

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid involved in multiple physiological processes. Importantly, dysregulated S1P levels are associated with several pathologies, including cardiovascular and inflammatory diseases and cancer. This report describes the successful production and characterization of a murine monoclonal antibody, LT1002, directed against S1P, using novel immunization and screening methods applied to bioactive lipids. We also report the successful generation of LT1009, the humanized variant of LT1002, for potential clinical use. Both LT1002 and LT1009 have high affinity and specificity for S1P and do not cross-react with structurally related lipids. Using an in vitro bioassay, LT1002 and LT1009 were effective in blocking S1P-mediated release of the pro-angiogenic and prometastatic cytokine, interleukin-8, from human ovarian carcinoma cells, showing that both antibodies can out-compete S1P receptors in binding to S1P. In vivo anti-angiogenic activity of all antibody variants was demonstrated using the murine choroidal neovascularization model. Importantly, intravenous administration of the antibodies showed a marked effect on lymphocyte trafficking. The resulting lead candidate, LT1009, has been formulated for Phase 1 clinical trials in cancer and age-related macular degeneration. The anti-S1P antibody shows promise as a novel, first-in-class therapeutic acting as a "molecular sponge" to selectively deplete S1P from blood and other compartments where pathological S1P levels have been implicated in disease progression or in disorders where immune modulation may be beneficial.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Cell Line, Tumor; Cell Proliferation; Choroidal Neovascularization; Cross Reactions; Female; Humans; Immunoglobulin Heavy Chains; Interleukins; Kinetics; Lysophospholipids; Macular Degeneration; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, SCID; Mutagenesis; Ovarian Neoplasms; Protein Binding; Signal Transduction; Sphingosine; Surface Plasmon Resonance

We previously demonstrated that sphingosine 1-phosphate (S1P) bimodally regulates epithelial ovarian cancer (EOC) cell invasiveness: low-concentration S1P stimulates invasion similar to lysophophatidic acid (LPA), while high-concentration S1P inhibits invasion. In this study, we investigated the mechanisms through which S1P affects EOC cell proteolysis, invasion, and adhesion in two cultured epithelial ovarian cancer cell lines.. G-protein Gi was inhibited by pertussis toxin (PTX) and GTP binding protein Rac by NSC23766. S1P conditioned media of DOV13 and OVCA429 cells were evaluated via gel zymography, fluorometric gelatinase assay, urokinase plasminogen activator (uPA) activity assay, and Western Blot for MT1-MMP. Cell invasion was analyzed in Matrigel chambers. Membrane-N-cadherin was localized via fluorescence microscopy.. Zymography revealed pro-MMP2 in conditioned media of EOC cells regardless of treatment. Gelatinase activity was increased by low-concentration S1P. In DOV13 cells this effect was Gi and Rac dependent. In all OVCA429 and control DOV13 cells, PTX enhanced gelatinolysis, suggesting an MMP2-inhibitory pathway via Gi. MT1-MMP was decreased Gi-dependently by high-concentration S1P. Rac inhibition significantly counteracted low-S1P enhancement and high-S1P reduction of DOV13 invasiveness; and uPA activity in conditioned media of invading cells correlated significantly. Immunohistochemistry revealed Gi-dependent clustering of membrane-N-cadherin in DOV13 cells treated with 0.5 microM S1P or 10 microM LPA.. S1P influences EOC invasion by regulating ECM-proteolysis and cell-cell attachment via MMP2, uPA, and membrane-N-cadherin. Furthermore, this study illustrates that the net effect of S1P on each of these processes reflects a complex interplay of multiple GPCR pathways involving Gi and downstream Rac.

Topics: Antigens, CD; Cadherins; Cell Adhesion; Cell Line, Tumor; Female; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Lysophospholipids; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Ovarian Neoplasms; Pertussis Toxin; rac GTP-Binding Proteins; Sphingosine; Urokinase-Type Plasminogen Activator

Epithelial ovarian cancer (EOC) arises from the epithelial layer covering the surface of ovaries and i.p. metastasis is commonly observed at diagnosis. Sphingosine-1-phosphate (S1P), a bioactive lipid signaling molecule, is potentially involved in EOC tumorigenesis. We have found that S1P is elevated in human EOC ascites. We show that physiologically relevant concentrations of S1P stimulate migration and invasion of EOC cells but inhibit migration of human ovarian surface epithelial (HOSE) cells. In addition, S1P inhibits lysophosphatidic acid (LPA)-induced cell migration in HOSE but not in EOC cells. We have provided the first line of evidence that the expression levels of S1P receptor subtypes are not the only determinants for how cells respond to S1P. Although S1P(1) is expressed and functional in HOSE cells, the inhibitory effect mediated by S1P(2) is dominant in those cells. The cellular preexisting stress fibers are also important determinants for the migratory response to S1P. Differential S1P-induced morphology changes are noted in EOC and HOSE cells. Preexisting stress fibers in HOSE cells are further enhanced by S1P treatment, resulting in the negative migratory response to S1P. By contrast, EOC cells lost stress fibers and S1P treatment induces filopodium-like structures at cell edges, which correlates with increased cell motility. In addition, inhibition of the protein kinase C pathway is likely to be involved in the inhibitory effect of S1P on LPA-induced cell migration in HOSE cells. These findings are important for the development of new therapeutics targeting S1P and LPA in EOC.

Topics: Ascites; Cell Line, Tumor; Cell Movement; Cell Shape; Enzyme Activation; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Lysophospholipids; Mitogen-Activated Protein Kinase Kinases; Ovarian Neoplasms; Protein Kinase C; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Stress Fibers

OVCAR3 ovarian cancer cells express three sphingosine 1-phosphate (S1P) receptors, S1P(1), S1P(2), and S1P(3), but not S1P(4). Stimulation of OVCAR3 cells with S1P induced intracellular calcium increases, which were partly inhibited by VPC 23019 (an S1P(1/3) antagonist). S1P-induced calcium increases were mediated by phospholipase C and pertussis toxin (PTX)-sensitive G-proteins in OVCAR3 cells. S1P stimulated extracellular signal-regulated kinase, p38 kinase, and Akt which were inhibited by PTX. S1P-stimulated chemotactic migration of OVCAR3 cells in a PTX-sensitive manner, indicating crucial role of G(i) protein(s) in the process. S1P-induced chemotactic migration of OVCAR3 cells was completely inhibited by LY294002 and SB203580. Pretreatment of VPC 23019 (an S1P(1/3) antagonist) completely inhibited S1P-induced chemotaxis. S1P also induced invasion of OVCAR3 cells, which was also inhibited by VPC 23019. Taken together, this study suggests that S1P stimulate chemotactic migration and cellular invasion, and VPC 23019-sensitive S1P receptor(s) might be involved in the processes.

Topics: Blotting, Western; Calcium; Cell Line, Tumor; Cell Movement; Chemotaxis; Chromones; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Lysophospholipids; Morpholines; Neoplasm Invasiveness; Ovarian Neoplasms; p38 Mitogen-Activated Protein Kinases; Pertussis Toxin; Phosphoinositide-3 Kinase Inhibitors; Protein Isoforms; Proto-Oncogene Proteins c-akt; Pyridines; Receptors, Lysosphingolipid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sphingosine

We previously demonstrated the regulation of epithelial ovarian cancer (EOC) cell invasiveness by the bioactive phospholipid sphingosine 1-phosphate (S1P). Low-dose S1P stimulated invasion like lysophosphatidic acid (LPA), while high-dose S1P inhibited invasion. Here we investigate how cell attachment status affects response to S1P and examine the effects of S1P and LPA on cell-cell and cell-extracellular matrix (ECM) adhesion.. EOC Dov13 cell invasion, ECM attachment and cell adhesion were tested through in vitro assays of Matrigel invasion and attachment to Matrigel, collagen or cell monolayer. Fractionated membrane and cytoplasmic proteins and biotin-labeled surface proteins were analyzed by western analysis. Actin cytoskeleton and FAK were visualized by immunofluorescence.. S1P (20 muM) inhibited invasion of sustained, attached cells but enhanced that of invading cells. Membrane N-cadherin was depleted upon reattachment to ECM. S1P pretreatment (20 muM) accelerated N-cadherin recovery, while 40 muM LPA or 0.5 muM S1P delayed recovery. Cell-cell adhesion and stress fibers were decreased by LPA and by 0.5 muM S1P but increased by 20 muM S1P. While S1P increased cellular attachment to Matrigel and collagen-I, LPA inhibited attachment to Matrigel. Surface N-cadherin, gamma- and beta-catenins, FAK and integrinbeta1 were altered by both reattachment and treatment with S1P or LPA.. S1P inversely affects invasion of attached and invading cells, switching from inhibition to stimulation. This switch is associated with depletion of N-cadherin and membrane FAK. The recovery of membrane N-cadherin, change in cell-cell adhesion and actin stress fibers intensity in response to LPA and S1P inversely correlate with their effects on cellular invasiveness.

Topics: Actins; Biocompatible Materials; Blotting, Western; Cadherins; Carcinoma; Cell Adhesion; Cell Line, Tumor; Collagen; Drug Combinations; Extracellular Matrix; Female; Fluorescent Antibody Technique; Focal Adhesion Kinase 1; Humans; Laminin; Lysophospholipids; Neoplasm Invasiveness; Ovarian Neoplasms; Proteoglycans; Sphingosine

Within the tumor microenvironment the invasiveness of epithelial ovarian carcinoma (EOC) cells is stimulated by biologically active lipids such as lysophosphatidic acid (LPA). We tested the in vitro effect of another bioactive lysophospholipid, sphingosine-1-phosphate (S1P), on the invasiveness of EOC cells.. Dov13 EOC cells were tested for invasion through matrigel-coated chambers and for gelatinase activity using a fluorogenic assay. cDNA was analyzed through real-time PCR. Cell surface proteins, isolated through biotinylation and affinity purification, were analyzed by Western blots.. Invasion of Dov13 cells was enhanced by low (0.5 microM) and inhibited by high (20 microM) concentrations of S1P, which correlated with increased and reduced gelatinase activity in conditioned media. Low and high S1P dose also differently affected the presentation of surface S1P receptors; low S1P dose increased S1P1 and decreased S1P2, while high S1P increased S1P3. LPA and S1P differently altered transcript levels of their respective and reciprocal receptors; receptors that were upregulated by one lysophospholipid (S1P2,3 and LPA1 by LPA, LPA3,4 and S1P1,4,5 by S1P) were downregulated or unchanged by the other.. The dual effect of high and low S1P concentration on invasion was probably caused by the diverse changes to the presentation of surface S1P receptors. The opposite effect of S1P and LPA on expression of each receptor suggests a homeostatic transcriptional mechanism that abrogates the effects of LPA and S1P on EOC cells. Altogether this study demonstrates a complex role of S1P in EOC cell invasion, a process highly balanced and regulated by LPA and S1P within the tumor microenvironment.

Topics: Dose-Response Relationship, Drug; Female; Humans; In Vitro Techniques; Lysophospholipids; Neoplasm Invasiveness; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Sphingosine; Tumor Cells, Cultured

Akt plays a pivotal role in cell survival and tumorigenesis. We investigated the potential interaction between sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF) in the Akt signaling pathway. Using mouse embryonic fibroblasts (MEFs) from S1P receptor knockout mice, we show here that S1P3 was required for S473 phosphorylation of Akt by S1P. In addition, S1P-stimulated activation of Akt, but not ERK, was blocked by a PDGF receptor (PDGFR)-specific inhibitor, AG1296, suggesting a S1P3-mediated specific crosstalk between the Akt signaling pathways of S1P and PDGFR in MEFs. We investigated this crosstalk under different conditions and found that both Akt and ERK activation induced by S1P, but not lysophosphatidic acid (LPA), in HEY ovarian cancer cells required PDGFR but not epidermal growth factor receptor (EGFR) or insulin-like growth factor-I receptor (IGFR). Importantly, S1P induced a Gi-dependent tyrosine phosphorylation of PDGFR in HEY cells. This dependence on PDGFR in S1P-induced Akt activation was also observed in A2780, T47D, and HMEC-1 cells (which express S1P3), but not in PC-3 or GI-101A cells (which do not express S1P3), further supporting that S1P3 mediates the crosstalk between S1P and PDGFR. This is the first report demonstrating a unique interaction between S1P3 and PDGFR, in addition to demonstrating a specific role for S1P3 in S1P-induced Akt activation.

Topics: Animals; Cell Line; Cell Line, Tumor; Enzyme Activation; Female; Fibroblasts; Humans; Lysophospholipids; Mice; Mitogen-Activated Protein Kinases; Organ Specificity; Ovarian Neoplasms; Phosphorylation; Phosphotyrosine; Platelet-Derived Growth Factor; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Sphingosine

To determine whether lysophosphatidic acid (LPA) and other lysophospholipids (LPL) are useful markers for diagnosis and/or prognosis of ovarian cancer in a controlled setting.. Plasma samples were collected from ovarian cancer patients and healthy control women in Hillsborough and Pinellas counties, Florida, and processed at the University of South Florida H. Lee Moffitt Cancer Center and Research Institute (Moffitt). Case patients with epithelial ovarian cancer (n = 117) and healthy control subjects (n = 27) participated in the study. Blinded LPL analysis, including 23 individual LPL species, was performed at the Cleveland Clinic Foundation using an electrospray ionization mass spectrometry-based method. LPL levels were transmitted to Moffitt, where clinical data were reviewed and statistical analyses were performed.. There were statistically significant differences between preoperative case samples (n = 45) and control samples (n = 27) in the mean levels of total LPA, total lysophosphatidylinositol (LPI), sphingosine-1-phosphate (S1P), and individual LPA species as well as the combination of several LPL species. The combination of 16:0-LPA and 20:4-LPA yielded the best discrimination between preoperative case samples and control samples, with 93.1% correct classification, 91.1% sensitivity, and 96.3% specificity. In 22 cases with both preoperative and postoperative samples, the postoperative levels of several LPL, including S1P, total LPA, and lysophosphatidylcholine (LPC) levels and some individual species of LPA and LPC, were significantly different from preoperative levels.. LPA, LPI, LPC, and S1P appear useful as diagnostic and prognostic biomarkers of ovarian cancer.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Female; Humans; Lysophospholipids; Middle Aged; Neoplasm Staging; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Peritoneal Neoplasms; Spectrometry, Mass, Electrospray Ionization; Sphingosine

Serum contains a variety of biomolecules, which play an important role in cell proliferation and survival. We sought to identify the serum factor responsible for mitigating tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis and to investigate its molecular mechanism. TRAIL induced effective apoptosis without serum, whereas bovine serum decreased apoptosis by suppressing cytochrome c release and caspase activation. Indeed, albumin-bound lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) inhibited TRAIL-induced apoptosis by suppressing caspase activation and cytochrome c release. LPA increased phosphatidylinositol 3-kinase (PI3K)-dependent Akt activation, cellular FLICE-inhibitory protein (cFLIP) expression, and Bad phosphorylation, resulting in inhibition of caspase-8 activation and Bad translocation to mitochondria. The antiapoptotic effect of LPA was abrogated by PI3K inhibitor, transfection with dominant-negative Akt, and specific downregulation of cFLIP expression using siRNA and further increased by siRNA-mediated suppression of Bad expression. Moreover, sera from ovarian cancer patients showed more protective effect against TRAIL-induced apoptosis than those from healthy donors, and this protection was suppressed by PI3K inhibitor. Our results indicate that albumin-bound LPA and S1P prevent TRAIL-induced apoptosis by upregulation of cFLIP expression and in part by Bad phosphorylation, through the activation of PI3K/Akt pathway.

Topics: Adult; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-Associated Death Protein; Carrier Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspases; Cattle; Cell Line, Tumor; Clone Cells; Down-Regulation; Drug Resistance, Neoplasm; Female; Humans; Intracellular Signaling Peptides and Proteins; Lysophospholipids; Male; Membrane Glycoproteins; Middle Aged; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Sphingosine; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Up-Regulation

We have previously described that bioactive lysophospholipids-lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC)-are present in ascitic fluids from patients with ovarian cancer. To understand the role of these lipids in ovarian cancer, we investigated the effects of these lipids on interleukin-8 (IL-8) production in ovarian cancer cells. IL-8 is a proinflammatory and proangiogenic factor, which is potentially involved in ovarian cancer development.. The Clontech PCR-Select cDNA subtraction method (Clontech Laboratories, Inc., Palo Alto, CA) was used to identify genes potentially regulated by LPA in HEY and OCC1 ovarian cancer cell lines. Northern blot analysis was used to confirm and examine IL-8 mRNA regulation by lysolipids. Enzyme-linked immunosorbent assay (ELISA) was used for detecting secreted IL-8.. We describe here that LPA, S1P, and SPC increased mRNA levels (2- to 7-fold) and protein secretion (2- to 12-fold) of IL-8 from ovarian cancer cells (HEY, OCC1, and SKOV3) in vitro. These regulations were both dose- and time-dependent. All three lipids increased the stability IL-8 mRNA in HEY cells. In contrast to malignant ovarian cancer cells, immortalized human ovarian epithelial cells did not respond to any of these lipids to increase the secretion of IL-8, although these cells secreted similar basal levels of IL-8 (310 pg/ml/10,000 cells). Two breast cancer cell lines (MCF7 and T47D) secreted lower basal levels of IL-8 (48-80 pg/ml/10,000 cells), compared with ovarian cancer cells (200-500 pg/ml/10,000 cells). MCF7 cells responded to LPA, but not S1P and SPC, by increasing the secretion of IL-8. T47D and MCF10A, an immortalized breast cell line, did not respond to LPA, S1P, or SPC to increase IL-8 secretion.. LPA, S1P, and SPC regulate the mRNA and protein levels of the proinflammatory and proangiogenic factor IL-8 in ovarian cancer cells. The pathological significance of these regulations in ovarian cancer remains to be further investigated.

Topics: Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Lysophospholipids; Ovarian Neoplasms; Phosphorylcholine; RNA, Messenger; Sphingosine; Tumor Cells, Cultured; Up-Regulation

Endothelial differentiation gene (edg)-encoded G protein-coupled receptors (Edg Rs)-1, -3, and -5 bind sphingosine 1-phosphate (S1P), and Edg-2 and -4 bind lysophosphatidic acid (LPA). Edg Rs transduce signals from LPA and S1P that stimulate ras- and rho-dependent cellular proliferation, enhance cellular survival, and suppress apoptosis. That high levels of LPA in plasma and ascitic fluid of patients with ovarian cancer correlate with widespread invasion suggested the importance of investigating expression and functions of Edg Rs in ovarian cancer cells (OCCs) as compared with nonmalignant ovarian surface epithelial cells (OSEs). Analyses of Edg Rs by semiquantitative reverse transcription-PCR, a radioactively quantified variant of PCR, and Western blots developed with monoclonal antibodies showed prominent expression of Edg-4 R in primary cultures and established lines of OCCs but none in OSEs. In contrast, levels of Edg-2, -3, and -5 were higher in OSEs than OCCs. LPA stimulated proliferation and signaled a serum response element-luciferase reporter of immediate-early gene activation in OCCs but not OSEs, whereas S1P evoked similar responses in both OSEs and OCCs. Pharmacological inhibitors of Edg R signaling suppressed OCC responses to LPA. A combination of monoclonal anti-Edg-4 R antibody and phorbol myristate acetate, which were inactive separately, evoked proliferative and serum response element-luciferase responses of OCCs but not OSEs. Thus the Edg-4 R may represent a distinctive marker of OCC that transduces growth-promoting signals from the high local concentrations of LPA characteristic of aggressive ovarian cancer.

Topics: Female; Humans; Insulin-Like Growth Factor II; Lysophospholipids; Nuclear Proteins; Ovarian Neoplasms; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Response Elements; RNA, Messenger; Signal Transduction; Sphingosine; Tetradecanoylphorbol Acetate; Transcription Factors

Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule. It stimulates the growth of some cells, but inhibits the growth of others. In this study, we describe the detection of sub-microM to microM concentrations of S1P in the ascitic fluids of patients with ovarian cancer. In ovarian cancer cells cultured in vitro, S1P exhibited a dual effect on growth and/or survival. S1P (10 microM) induced cell death when cells were in suspension but stimulated cell growth when cells were attached. The calcium-dependent induction of cell death by S1P is apparently associated with its inhibitory effect on cell attachment and cell adhesion. S1P (10-30 microM) also induced calcium-dependent cell-cell aggregation.

Topics: Ascites; Cell Adhesion; Cell Aggregation; Cell Death; Cell Division; Female; Humans; Lysophospholipids; Ovarian Neoplasms; Sphingosine; Tumor Cells, Cultured