No abstract

Disruptions of cell death signalling occur in pathological processes, such as cancer and degenerative disease. Increased knowledge of cell death signalling has opened new areas of therapeutic research, and identifying key mediators of cell death has become increasingly important. Early triggering events in cell death may provide potential therapeutic targets, whereas agents affecting later signals may be more palliative in nature. A group of primary mediators are derivatives of the highly unsaturated fatty acids (HUFAs), particularly oxygenated metabolites such as prostaglandins. HUFAs, esterified in cell membranes, act as critical signalling molecules in many pathological processes. Currently, agents affecting HUFA metabolism are widely prescribed in diseases involving disordered cell death signalling. However, partly due to rapid metabolism, their role in cell death signalling pathways is poorly characterized. Recently, HUFA-derived mediators, the resolvins/protectins and endocannabinoids, have added opportunities to target selective signals and pathways. This review will focus on the control of cell death by HUFA, eicosanoid (C20 fatty acid metabolites) and docosanoid (C22 metabolites), HUFA-derived lipid mediators, signalling elements in the micro-environment and their potential therapeutic applications. Further therapeutic approaches will involve cell and molecular biology, the multiple hit theory of disease progression and analysis of system plasticity. Advances in the cell biology of eicosanoid and docosanoid metabolism, together with structure/function analysis of HUFA-derived mediators, will be useful in developing therapeutic agents in pathologies characterized by alterations in cell death signalling. AbbreviationsDHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; NSAID, nonsteroidal anti-inflammatory drug; PG, prostaglandin; AA, arachidonic acid; HUFA, highly unsaturated fatty acids with 4 or more bonds, for example, arachidonic, eicosapentaenoic and docosahexaenoic acids (4, 5 and 6 double bonds respectively); PUFA, polyunsaturated fatty acids, with 2 or more unsaturated C-C bonds; HUFA, highly unsaturated C20 fatty acid, with 3 or more unsaturated C-C bonds Many therapeutic agents influence cell death signalling and highly unsaturated fatty acid (HUFA) metabolism (Figure 1). These agents may act at the level of metabolic events affecting apoptosis, enzyme systems and cofactors, agents affecting cell cycle progression and DNA repair, and oncogene expression. Intracellularly, agents affecting organelles and the mitochondrial intrinsic pathway, endoplasmic reticulumassociated stress pathways and lysosomal autophagy can have profound effects on cell death. There has also been development of agents affecting transcellular signalling via the extrinsic pathway, oxidative stress, growth factors and lipid mediators, ion and metabolite flux, adhesion and migration.

The prognosis for patients with malignant gliomas is poor, but improvements may emerge from a better understanding of the pathophysiology of glioma signalling. Recent therapeutic developments have implicated lipid signalling in glioma cell death. Stress signalling in glioma cell death involves mitochondria and endoplasmic reticulum. Lipid mediators also signal via extrinsic pathways in glioma cell proliferation, migration and interaction with endothelial and microglial cells. Glioma cell death and tumour regression have been reported using polyunsaturated fatty acids in animal models, human ex vivo explants, glioma cell preparations and in clinical case reports involving intratumoral infusion. Cell death signalling was associated with generation of reactive oxygen intermediates and mitochondrial and other signalling pathways. In this review, evidence for mitochondrial responses to stress signals, including polyunsaturated fatty acids, peroxidizing agents and calcium is presented. Additionally, evidence for interaction of glioma cells with primary brain endothelial cells is described, modulating human glioma peroxidative signalling. Glioma responses to potential therapeutic agents should be analysed in systems reflecting tumour connectivity and CNS structural and functional integrity. Future insights may also be derived from studies of signalling in glioma-derived tumour stem cells.

Sphingosine I-phosphate (SIP), lysophosphatidic acid (LPA) and phosphatidic acid (PA) bind to Gi/o-protein coupled receptors (GPCRs) to stimulate the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathway human embyronic kidney 293 (HEK293) cells. The bioavailability of these agonists at their receptors may be limited by a family of lipid phosphate phosphatases (LPP1, la, 2 and 3) which catalyse the dephosphorylation of these phosphorylated lipids. However, this study shows that although the stimulation of p42/p44 MAPK by SlP, LPA and PA was substantially reduced in cells transfected with LPPI, l a and 2, this was correlated with reduced basal intracellular phosphatidic acid and not ecto-LPP activity. Additionally, LPPl, l a and 2 also inhibited the stimulation of p42/p44 MAPK by thrombin, a peptide GPCR agonist that is not an LPP substrate. Since the LPPs had no effect on the stimulation of p42/p44 MAPK by other agents that d o not use G-proteins to signal, such as serum factors and phorbol ester, these findings suggest that LPPl, l a and 2 may function to perturb GPCR signalling per se. This may involve inhibition of receptor-mediated endocytosis, which is often required for GPCR internalisation and p42/p44 MAPK activation.We previously demonstrated that the signalling molecule arachidonic acid (AA), induced inhibition of cell growth and apoptosis in bcr-abl transformed leukemia cell line, H7.A54.bcr/abl and haematopoietic progenitor cells from patients with chronic myeloid leukemia (Cancer Res. 59:5047, 1999). The cell-type specificity of AA associated antiproliferative activities was investigated. Dosedependent inhibition of cell growth was detected on incubation of H7.A54.bcr/abl, HL-60 (human promyelocytic leukemia), JURKAT (human acute T-cell leukemia) and U937 (human histiocytic lymphoma) cell lines with 10-100pM AA. AA (100pM,72h) inhibited growth of H7.A54.bcr/abl, HL-60, JURKAT and U937 cell lines by 98%,53%,68%,20% respectively. In contrast, 10-100pM AA had no effect on RPMI 7666 (human normal lymphoblast)cell growth. AA effects on two kinases implicated in leukemic cell apoptosis: P38 mitogen activated protein kinase, p38-MAPK and c-jun amino-terminal kinase UNK) were investigated. J N K activation was detected in H7.A54 cells incubated with 10-lOOpM AA. J N K was also activated by AA in JURKAT, U937, RPMI 7666 cells but not in HL-60 cells. Additionally, AA activated p38-MAPK in H7.A54 bcr/abl, HL-60, U937 cells but not JURKAT and RPI 7666 cells. These results suggest inhibition of cell growth and activation of p38-MAPK and J N K by AA is type-specific. Moreover, leukemic cells were more sensitive to AA inhibitorv effects than normal cells.36 Vavl is required for platelet aggregation to threshold concentrations of Glycoprotein VI agonist, independent of phospholipase C and RacThe GDP/GTP exchange factor Vavl has been shown to have an important role in T-cell receptor signalling, a pathway that has similar components to that of the collagen receptor, Glycoprotein VI (GPVI), in platelets. In t...

Prostaglandin E2 (PGE2) plays a crucial role in angiogenesis as well as in ischemic and inflammatory disorders of the brain associated with breakdown of the blood-brain barrier. However, the effects of PGE2 on brain endothelial cell migration, a key process in the angiogenic response and blood-brain barrier stability, are not well defined. Exposure of human brain endothelial cells (HBECs) to PGE2 elicited a chemotactic response in a time- and dose-dependent manner. The maximum migratory response was detected following 8 hours exposure of HBECs to PGE2 (100 nM). Migration of HBECs in response to PGE2 was accompanied by profound changes in the reorganization of actin filaments. Fluorescence microscopy examination of NBD-phallacidin-labeled endothelial cells showed increased formation of stress fibers, lamellipodia and podosomes after treatment with PGE2 (100 nM) compared to control. Based on these results, we hypothesized that Rho-kinase (ROCK), an enzyme involved in regulation of actin dynamics and cell migration, mediated the effects of PGE2 on HBEC migration. Western blot analyses revealed that ROCK II (type alpha), but not ROCK I (type beta), was expressed in HBECs. To examine ROCK II activation, we performed immunocomplex kinase assays using myosin light chain (MLC) as a substrate. PGE2 (100 nM) induced a 2-fold increase of 32P-incorporation into MLC indicating activation of ROCK II. Pretreatment of HBECs with the selective ROCK inhibitor, Y27632 (150 nM), blunted HBEC migration in response to PGE2 but had no effect on migration induced by fetal bovine serum (10%). Knockdown of ROCK II by siRNA also abrogated the migratory response of HBECs to PGE2. In contrast, similar treatment had no effect of HBEC migration stimulated by hepatocyte growth factor. Taken together, these results are consistent with the hypothesis that stimulation of HBECs with PGE2 leads to activation of ROCK II, reorganization of the actin cytoskeleton and ultimately migration. A better characterization of the molecular events that regulate migration of brain endothelial cells is critical for the development of novel strategies to treat cerebrovascular diseases associated with deregulated angiogenesis.