T-oligos inhibit growth and induce apoptosis in human ovarian cancer cells

Ovarian cancer remains a leading cause of death among women worldwide, and current treatment regimens for advanced disease are inadequate. Oligonucleotides with sequence homology to telomeres (called T-oligos) have been shown to mimic DNA damage responses in cells and induce cytotoxic effects in certain tumor cell lines. We studied the effects of 2 distinct 16 mer T-oligos in 4 human ovarian epithelial carcinoma cell lines. A T-oligo with perfect homology to the telomere overhang region demonstrated some cytotoxic activity in half of the cell lines. A G-rich T-oligo derivative showed more potency and broader cytotoxic activity in these lines than the parental T-oligo. Activation of apoptotic pathways in ovarian cancer cells by exposure to the T-oligo was demonstrated by multiple independent assays. T-oligo was shown to have additive, or more than additive, activity in combination with 2 different histone deacetylase drugs currently in clinical testing. T-oligos may therefore provide a new and tumor-targeted approach to ovarian cancers.     

Sphingosine-1-phosphate modulates growth and adhesion of ovarian cancer cells

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.     

NSAIDs induce apoptosis in nonproliferating ovarian cancer cells and inhibit tumor growth in vivo

Ovarian cancer (OC) is one of the most lethal gynaecological cancers, which usually has a poor prognosis due to late diagnosis. A large percentage of the OC cell population is in a nonproliferating and quiescent stage, which poses a barrier to success when using most chemotherapeutic agents. Recent studies have shown that several nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in the treatment of OC. Furthermore, we have previously described the molecular mechanisms of NSAIDs' induction of cancer apoptosis. In this report, we evaluated various structurally distinct NSAIDs for their efficacies in inducing apoptosis in nonproliferating OC cells. Although several NSAIDs-induced apoptosis, Flufenamic Acid, Flurbiprofen, Finasteride, Celocoxib, and Ibuprofen were the most potent NSAIDs inducing apoptosis. A combination of these agents resulted in an enhanced effect. Furthermore, we demonstrate that the combination of Flurbiprofen, which targets nonproliferative cells, and Sulindac Sulfide, that affects proliferative cells, strongly reduced tumor growth when compared with a single agent treatment. Our data strongly support the hypothesis that drug treatment regimens that target nonproliferating and proliferating cells may have significant efficacy against OC. These results also provide a rationale for employing compounds or even chemically modified NSAIDs, which selectively and efficiently induce apoptosis in cells during different stages of the cell cycle, to design more potent anticancer drugs.     

P2X7 receptors induce degranulation in human mast cells

Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation. This raises the possibility that calcium influx through P2X receptors may lead to antigen-independent secretion of histamine and other granule-derived mediators from human mast cells. Here we show that activation of P2X7 receptors with both ATP and BzATP induces robust calcium rises in human mast cells and triggers their degranulation; both effects are blocked by the P2X7 antagonist AZ11645373, or the removal of calcium from the extracellular medium. Activation of P2X1 receptors with αβmeATP also induces calcium influx in human mast cells, which is significantly reduced by both PPADS and NF 449. P2X1 receptor activation, however, does not trigger degranulation. The results indicate that P2X7 receptors may play a significant role in contributing to the unwanted activation of mast cells in chronic inflammatory conditions where extracellular ATP levels are elevated.     

Nanosecond electric pulses induce DNA breaks in cisplatin-sensitive and -resistant human ovarian cancer cells

Human ovarian cancer cells COC1 and COC1/DDP (cisplatin-resistant subline) were exposed to 6 kV/cm nanosecond electric pulses (nsEP) with a pulse length of 8, 16 or 24 ns. The potential in a subcellular unit was calculated using a multilayer dielectric spherical model, and area under the voltage–time curves (AUC) integrated with a lower limit of 0.2 V. Cell viability was determined, and double-stand and total DNA breaks detected with the neutral and alkaline comet assays. nsEP evoked a higher voltage and AUC in nucleoplasm, and the levels in COC1 cells was just above those in COC1/DDP cells. Comets only appeared in the alkaline assay demonstrating single-stand DNA break. Fewer DNA break (16.51% vs. 35.13% at 24 ns, p = 0.0150) and more survival (22.42% vs. 13.19% at 24 ns, p = 0.0015) occurred in COC1/DDP cells despite an equal electric energy and almost equal cell sizes. 24-ns EP led to higher rates of cell-death and comet. The comet rate correlated with cell-death fraction in either cell line (r = 0.5701, p = 0.0135; r = 0.5110, p = 0.0302). There was no a correlation between the tail length, tail moment or Olive tail moment and cell-death rate. The data showed that response of chemosensitive cells differed from that of chemoresistant cells and DNA damage contributed to percent of cell death.     

Resveratrol downregulates Akt/GSK and ERK signalling pathways in OVCAR-3 ovarian cancer cells

Phytochemicals constitute a heterogeneous group of substances with an evident role in human health. Their properties on cancer initiation, promotion and progression are well documented. Particular attention is now devoted to better understand the molecular basis of their anticancer action. In the present work, we studied the effect of resveratrol on the ovarian cancer cell line OVCAR-3 by a proteomic approach. Our findings demonstrate that resveratrol down-regulates the protein cyclin D1 and, in a concentration dependent manner, the phosphorylation levels of protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β). The dephosphorylation of these kinases could be responsible for the decreased cyclin D1 levels observed after treatment. We also showed that resveratrol reduces phosphorylation levels of the extracellular signal-regulated kinase (ERK) 1/2. Chemical inhibitors of phosphatidylinositol 3-kinase (PI3K) and ERK both increased the in vitro therapeutic efficacy of resveratrol. Moreover, resveratrol had an inhibitory effect on the AKT phosphorylation in cultured cells derived from the ascites of ovarian cancer patients and in a panel of human cancer cell lines. Thus, resveratrol shows antitumor activity in human ovarian cancer cell lines targeting signalling pathway involved in cell proliferation and drug-resistance.     

Epidermal Notch signalling: differentiation, cancer and adhesion

The Notch pathway plays an important role in regulating epidermal differentiation. Notch ligands, receptors and effectors are expressed in a complex and dynamic pattern in embryonic and adult skin. Genetic ablation or activation of the pathway reveals that Notch signalling promotes differentiation of the hair follicle, sebaceous gland and interfollicular epidermal lineages and that Notch acts as an epidermal tumour suppressor. Notch signalling interacts with a range of other pathways to fulfil these functions and acts via RBP-Jkappa dependent and independent mechanisms. The effects on differentiation can be cell autonomous and non-autonomous, and Notch contributes to stem cell clustering via modulation of cell adhesion.     

Autocrine BMP4 signalling regulates ID3 proto-oncogene expression in human ovarian cancer cells

Bone morphogenetic protein (BMP)-4 signalling leads to the direct upregulation of ID3 proto-oncogene expression in human ovarian cancer cells. An upstream BMP4-responsive enhancer element consisting of a palindromic BMP response element (BRE) site and CAGA box was identified ~3.0 kb upstream of the human ID3 gene, and a nearly-identical element exists in the second intron of the ID3 gene. BMP4 stimulation leads to the direct binding of Smads 1/5 and Smad4 to the upstream and intronic enhancers, and together both enhancers cooperate to yield heightened BMP4-mediated ID3 promoter activity. We further demonstrate that ID3 is overexpressed in human ovarian cancer cells when compared to normal ovarian surface epithelial cells, and treatment of ovarian cancer cells with the BMP4 antagonist Noggin abrogates endogenous ID3 gene expression. Our findings define the mechanism of BMP4-mediated ID3 gene expression, and support the notion that ovarian cancer cells possess autocrine BMP4 signalling required to sustain ID3 overexpression which may contribute to human ovarian cancer pathogenesis.     

Chronic increases in sphingosine kinase-1 activity induce a pro-inflammatory, pro-angiogenic phenotype in endothelial cells

Sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which has potent pro-inflammatory and pro-angiogenic effects. We investigated the effects of raised SK1 levels on endothelial cell function and the possibility that this signaling pathway is activated in rheumatoid arthritis. Human umbilical vein endothelial cells with 3- to 5-fold SK1 (EC^SK) overexpression were generated by adenoviral and retroviralmediated gene delivery. The activation state of these cells and their ability to undergo angiogenesis was determined. S1P was measured in synovial fluid from patients with RA and OA. EC^SK showed an enhanced migratory capacity and a stimulated rate of capillary tube formation. The cells showed constitutive activation as evidenced by the induction of basal VCAM-1 expression, and further showed a more augmented VCAM-1 and E selectin response to TNF compared with empty vector control cells (EC^EV). These changes had functional consequences in terms of enhanced neutrophil binding in the basal and TNFstimulated states in EC^SK. By contrast, over-expression of a dominant-negative SK inhibited the TNF-induced VCAM-1 and E selectin and inhibited PMN adhesion, confirming that the observed effects were specifically mediated by SK. The synovial fluid levels of S1P were significantly higher in patients with RA than in those with OA. Small chronic increases in SK1 activity in the endothelial cells enhance the ability of the cells to support inflammation and undergo angiogenesis, and sensitize the cells to inflammatory cytokines. The SK1 signaling pathway is activated in RA, suggesting that manipulation of SK1 activity in diseases of aberrant inflammation and angiogenesis may be beneficial.     

Cell-cell adhesion and signalling

Signalling pathways activated by Rho small GTPases have recently been identified that coordinate junction assembly, stability and function, as well as interactions of adhesive complexes with the underlying cortical cytoskeleton. Particularly exciting is the interplay between adherens junctions, activation of Rho proteins and the dynamics of microtubule, actin and intermediate filaments. This interplay has important implications for functional regulation of cell-cell adhesion, and points to a more integrated view of signalling processes.     

Substance P induces degranulation of mast cells and leukocyte adhesion to venular endothelium

Substance P (SP), one of the established neurotransmitters, evokes an immunoinflammatory response involving leukocyte adhesion to venular endothelium and the degranulation of mast cells. The pathogenetic relationship between these responses, however, remains unresolved. In this study, we propose to examine the changes associated with the activation of mast cells, as well as leukocyte adhesion to venular endothelium by in vivo observation of the rat mesentery. The use of an in vitro assay for intracellular Ca²⁺ mobilization and the degranulation of mast cells demonstrated the significant upper shift of concentration response to SP (10⁻⁴–10⁻⁵ M). In vivo experiments on the mesenteric microcirculation also showed that SP induced a significant increase in the number of degranulated mast cells as well as in the number of leukocytes adherent to the venular wall. Tranilast, a mast cell stabilizer, as well as SP antagonist (CP-96,345) significantly attenuated the extent of mast cell degranulation and leukocyte adhesion elicited by SP. Although an immunoneutralization against CD18 by WT-3 significantly attenuated the leukocyte adhesion, it had no influence on the mast cell degranulation after SP superfusion. These separate in vivo observations show that SP induces leukocyte adhesion to the venular endothelium, possibly through the degranulation of mast cells.     

PTPN12 controls PTEN and the AKT signalling to FAK and HER2 in migrating ovarian cancer cells

CI-976 is a lysophospholipid acyltransferase antagonist that is known to affect secretory and endocytic membrane-trafficking pathways likely by increasing the lysophospholipid content in membranes. Our previous study suggested that lysophospholipids formed through the action of an intracellular phospholipase A₁, KIAA0725p (also known as DDHD2 and iPLA₁γ), may be important for the association of this enzyme with membranes. In this study, we examined the effect of CI-976 on the membrane association of KIAA0725p. While in HeLa cells KIAA0725p is localized in the Golgi and cytosol, in mouse embryonic fibroblasts (MEFs), it was found to be principally localized in the cytosol with some on post-endoplasmic reticulum compartments including the cis-Golgi. Treatment of MEFs with CI-976 induced the redistribution of KIAA0725p to membrane tubules, which were in vicinity to fragmented mitochondria. These tubules were not decorated with canonical organelle markers including Golgi proteins. A human KIAA0725p mutant, which exhibits decreased membrane-binding ability, was also redistributed to membrane structures upon CI-976 treatment. Our data suggest that the association of KIAA0725p with membranes is regulated by lipid metabolism, and that CI-976 may create unique membrane structures that can be marked by KIAA0725p.     

Paxillin and focal adhesion signalling

To facilitate a rapid response to environmental change, cells use scaffolding - or adaptor - proteins to recruit key components of their signal-transduction machinery to specific subcellular locations. Paxillin is a multi-domain adaptor found at the interface between the plasma membrane and the actin cytoskeleton. Here it provides a platform for the integration and processing of adhesion- and growth factor-related signals.     

Colon cancer cells: pro-invasive signalling

Colon cancer results from erroneous renewal of the enteric epithelium. Mutations in stem cells, or their proliferative progenitors, cause accumulation of cells that invade into the stroma and continue to divide rather than migrating on top of the basement membrane prior to entering into apoptosis. Many of these changes in invasive activity appear to be related to the invasion-suppressor role of E-cadherin. We have also investigated Listeria monocytogenes and other enteric bacteria, since these bacteria stimulate invasion through the production of a beta-casein-derived 13-amino acid peptide which is produced by enzymes present in the colon cancer ecosystem. The pro-invasive 13-amino acid peptide signals via small guanosine triphosphatases, which modulate the actin cytoskeleton, and via phosphorylation of the delta opioid receptor. The pro-invasive activity of this peptide is neutralized by the delta opioid receptor antagonist, naloxone. Since the delta opioid receptor belongs to the family of G protein-coupled receptors, implicated in colon cancer cell invasion signalling pathways, it is tempting to speculate that opioids could play a role in mediating this trait of malignant tumours.     

Interleukin-6 signaling regulates anchorage-independent growth, proliferation, adhesion and invasion in human ovarian cancer cells

It has been widely reported that Interleukin-6 (IL-6) is overexpressed in the serum and ascites of ovarian cancer (OVCA) patients, and elevated IL-6 level correlates with poor prognosis and survival. However, the exact role that IL-6 plays in this malignancy or whether IL-6 can regulate tumorigenic properties has not been established. Here we demonstrate that overexpression of IL-6 in non-IL-6-expressing A2780 cells (by transfecting with plasmid encoding for sense IL-6) increases anchorage-independent growth, proliferation, adhesion and invasion, while depletion of endogenous IL-6 expression in IL-6-overexpressing SKOV-3 cells (by transfecting with plasmid encoding for antisense IL-6) decreases. Further investigation indicates that IL-6 promotes OVCA cell proliferation by altering cell cycle distribution rather than inhibiting apoptosis and that IL-6-enhanced OVCA cell invasive may be associated with increased matrix metalloproteinase (MMP)-9 but not MMP-2 proteolytic activity. In addition, overexpressing or deleting of IL-6 in OVCA cells enhances or reduces its receptor (IL-6Rα and gp130) expression and basal phosphorylation levels of both ERK and Akt, and additional treatment with specific inhibitor of the ERK or Akt signaling pathway significantly inhibits the proliferation of IL-6-overexpressing A2780 cells. Our data suggest that the autocrine production of IL-6 by OVCA cells regulates tumorigenic properties of these cells by inducing IL-6 signaling pathways. Thus, regulation of IL-6 expression or its related signaling pathway may be a promising strategy for controlling the progression of OVCA.