The activity of arachidonic acid and gamma-linolenic acid on human gliomas

Despite recent advances in tumour cell biology, the prognosis for patients suffering from malignant glioma remains poor. Although primary glioma rarely metastasises outside the central nervous system (primary being defined as the mass of tumour cells at the original site of the neoplastic event) median survival of adults is less than 1 year after diagnosis.The efficacy of existing therapeutic interventions is limited by poor penetration of chemotherapeutic drugs across the blood brain barrier, the inherent radioresistance of glioma tissue and the infiltrating nature of the tumour. Further progress is likely to be achieved through analysis of the complex biology of these tumours and the development of novel therapeutic strategies. The purpose of this study was to investigate the therapeutic potential of the n-6 essential fatty acids arachidonic acid and gamma-linolenic acid, which may inhibit tumour proliferation by acting as substrates for the production of potentially cytotoxic reactive oxygen intermediates and stimulating apoptotic cell death, both alone and in conjunction with radiation.Experiments were undertaken to investigate the effects of exogenous arachidonic acid and gamma-linolenic acid on cellular peroxidation, proliferation, viability and apoptosis. These investigations were carried out on single cell suspensions of morphologically heterogeneous fresh human glioma tissue and associated normal brain, human phagocytes and the rat C6 glioma cell line. It was shown that oxidative activity was impaired in human glioma tissue. Addition of 4-40μM arachidonic acid and gamma-linolenic acid induced a concentration dependant increase in tumour reactive oxygen intermediate production and apoptotic activity. Although the kinetics of reactive oxygen intermediate formation in the presence of arachidonic acid and gamma-linolenic acid followed an exponential function in both normal and tumour cell preparations, tumour cells showed a significantly higher sensitivity to exogenous essential fatty acid stimulus. The kinetics of this stimulation were grade dependent, with high grade tumours responding in a more rapid and sustained manner in comparison with lower grade tumours. The morphological heterogeneity of the human glioma preparations was confirmed with immunohistochemical analysis and flow cytometry using monoclonal and polyclonal anti-Glial Acidic Fibrillary Protein (GFAP). GFAP positive cells responded to exogenous arachidonic acid and gamma-linolenic acid with increased reactive oxygen intermediate production, indicating a high sensitivity of glioma cells to essential fatty acid stimulus. Reactive oxygen intermediate production was also investigated in phagocyte preparations of patients undergoing pulmonary resection for lung cancer. It was found that reactive oxygen intermediate generation was stimulated in patient and control phagocytes by exogenous 1 -40μM arachidonic acid and gamma-linolenic acid both pre and post-operatively. Increased reactive oxygen intermediate formation was detected in the cell population identified as leukocytes in preparations of human primary glioma, although this response was less than that of associated tumour. It was also found that surgery was associated with an increase in phagocyte reactive oxygen intermediate at 2 and 7 days post-operatively in lung cancer patients. The interactive effects of arachidonic acid, gamma-linolenic acid and therapeutic radiation were demonstrated in the rat C6 glioma cell line. The rate ofreactive XVI oxygen intermediate production in response to exogenous arachidonic acid and gamma-linolenic acid increased within the first hour, and elevated oxidative activity was detected for up to three hours. However, a different pattern ofreactive oxygen intermediate generation was observed in response to radiation alone. Similarly, an early apoptotic response was observed following exogenous arachidonic acid and gamma-linolenic acid stimulation. In comparison, radiation induced stimulation of apoptosis occurred over the 12 hour period of incubation and was maximal between 6 and 8 hours post-irradiation. An enhanced radiation response was observed when the stimulation of apoptosis induced by essential fatty acid stimulus alone was low, suggesting that essential fatty acids and radiation may interact to potentiate reactive oxygen intermediate generation and apoptosis.In conclusion, this study has provided evidence that glioma tissue has low basal oxidative activity in comparison with associated normal brain, and that addition of exogenous arachidonic acid and gamma-linolenic acid stimulates peroxidative and apoptotic activity in glioma tissue a grade dependant manner. Studies on the cellular heterogeneity of human glioma samples indicate that the stimulation ofreactive oxygen intermediate production by exogenous arachidonic acid and gamma-linolenic acid occurs in GFAP positive cells. This indicates high sensitivity of human glioma to exogenous essential fatty acid stimulus. Phagocyte populations from lung cancer and malignant glioma patients also respond with increased reactive oxygen intermediate production to exogenous arachidonic acid and gamma-linolenic acid, although the magnitude of this increase is less than that observed for tumour cells. In addition, there is evidence ofpotentiation ofthe oxidative and apoptotic response of the rat C6 cell line to exogenous arachidonic acid and gamma-linolenic acid in the presence of therapeutically relevant doses ofradiation. These results are consistent with a clinical role for arachidonic acid and gamma-linolenic acid in the treatment of malignant glioma

ΦΥΤΟΧΗΜΙΚΗ ΜΕΛΕΤΗ ΤΟΥ ΦΥΤΟΥ: Hypericum trichocaulon Boiss. & Heldr. – Hypericaceae

Φυτοχημική ανάλυση του κυκλοεξανικού και μεθανολικού εκχυλίσματος του φυτού Hypericum trichocaulon, το οποίο ανήκει στα ενδημικά φυτά της Κρήτης.Eleven compounds were isolated from the aerial parts of Hypericum trichocaulon Boiss. & Heldr., growing wild in the island of Crete (Greece). These compounds comprise one novel prenylated phloroglycinol derivative, named adhyperfoliatin and ten known compounds, procyanidin A2, hyperixanthone A, (E)-chlorogenic acid, trans-phytol, five flavonoids, i.e. I3, II8-biapigenin, quercitrin, hyperoside, rutin, myricitrin and one fatty acid. The structures of the isolated compounds were established by means of NMR [1H-1H-COSY, 1H-13C-HSQC, HMBC, NOESY, ROESY] and MS spectral analyses

The modulatory effect of cytokines on cell proliferation in C6 glioma cells.

by Liu Heng.Thesis (M.Phil.)--Chinese University of Hong Kong, 1996.Includes bibliographical references (leaves 115-138).Acknowledgments --- p.IList of Abbreviations --- p.IIAbstract --- p.VChapter Chapter 1: --- IntroductionChapter 1.1 --- Cytokines in the Central Nervous System --- p.1Chapter 1.1.1 --- Basic Properties of Cytokines --- p.1Chapter 1.1.2 --- The General Characteristics of Glial Cells --- p.4Chapter 1.1.2.1 --- Astrocytes --- p.4Chapter 1.1.2.2 --- Oligodendrocytes --- p.6Chapter 1.1.2.3 --- Microglial --- p.7Chapter 1.1.3 --- The Effects of Cytokines on Neural Cells --- p.7Chapter 1.1.3.1 --- TNF-α and Neural Cells --- p.8Chapter 1.1.3.2 --- LIF and Neural Cells --- p.10Chapter 1.1.3.3 --- IL-1 and Neural Cells --- p.12Chapter 1.1.3.4 --- IL-6 and Neural Cells --- p.14Chapter 1.1.4 --- Immune Response in the Central Nervous System --- p.16Chapter 1.2 --- The C6 Glioma as a Model for the Study of Glial Cell Growth and Differentiation --- p.21Chapter 1.2.1 --- The Rat C6 Glioma Cells --- p.21Chapter 1.2.2 --- The Differentiation and Proliferation of C6 Glioma Cells --- p.23Chapter 1.3 --- Signal Transduction Pathways in Cytokine-stimulated Glial Cells --- p.28Chapter 1.3.1 --- Intracellular Signalling Pathways of Cytokines --- p.28Chapter 1.3.1.1 --- Protein Kinase C Pathway --- p.29Chapter 1.3.1.2 --- Tyrosine Kinase Pathway --- p.30Chapter 1.3.1.3 --- Cyclic Nucleotide Pathway --- p.32Chapter 1.3.1.4 --- Nitric Oxide Pathway --- p.33Chapter 1.3.2 --- Intracellular Signalling Pathways in Cytokine-stimulated C6 Glioma Cells --- p.34Chapter 1.4 --- The Aims of This Thesis Project --- p.37Chapter Chapter 2: --- Materials and Methods --- p.41Chapter 2.1 --- Rat C6 Glioma Cell Culture --- p.41Chapter 2.1.1 --- Preparation of Culture Media --- p.41Chapter 2.1.1.1 --- Complete Dulbecco's Modified Eagle Medium --- p.41Chapter 2.1.1.2 --- Complete Roswell Park Memorial Institute1640 Medium --- p.42Chapter 2.1.2 --- Maintenance of the C6 Cell Line --- p.42Chapter 2.1.3 --- Cell Preparation for Assays --- p.43Chapter 2.2 --- Determination of Cell Proliferation --- p.44Chapter 2.2.1 --- Determination of Cell Proliferation by [3H]-Thymidine Incorporation --- p.44Chapter 2.2.2 --- Measurement of Cell Viability Using Neutral Red Assay --- p.45Chapter 2.2.3 --- Data Analysis --- p.45Chapter 2.3 --- Effects of Cytokines and Lipopolysaccharide on C6 Cell Proliferation --- p.46Chapter 2.4 --- Effects of Protein Kinase C Activators and Inhibitors on Cytokine-induced C6 Cell Proliferation --- p.47Chapter 2.5 --- Effects of cAMP or cGMP on Cytokine-induced C6 Cell Proliferation --- p.48Chapter 2.6 --- Effects of Tyrosine Kinase Inhibitors on Cytokine-induced C6 Cell Proliferation --- p.48Chapter 2.7 --- Effects of Calcium Ion on Cytokine-induced C6 Cell Proliferation --- p.49Chapter 2.8 --- Effects of Nitric Oxide on Cytokine-induced C6 Cell Proliferation --- p.49Chapter 2.8.1 --- Effects of Sodium Nitroprusside and Nitric Oxide Synthase Inhibitors on Cytokine-induced C6 Cell Proliferation --- p.49Chapter 2.8.2 --- Nitric Oxide Production Assay --- p.50Chapter 2.9 --- Effects of β-Adrenergic Receptor Agonist and Antagonist on Cytokine-induced C6 Cell Proliferation --- p.51Chapter 2.10 --- Morphological Studies on Cytokine-Treated C6 Glioma Cells --- p.51Chapter 2.10.1 --- Wright-Giesma Staining --- p.52Chapter 2.10.2 --- Glial Fibrillary Acidic Protein Staining --- p.52Chapter 2.10.3 --- Hematoxylin Staining --- p.53Chapter Chapter 3: --- Results --- p.55Chapter 3.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.55Chapter 3.1.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.56Chapter 3.1.2 --- The Time Course of Cytokine-induced C6 Cell Proliferation --- p.59Chapter 3.1.3 --- Effects of Lipopolysaccharide on C6 Cell Proliferation --- p.61Chapter 3.1.4 --- Effects of Cytokines on the Growth of C6 Cells --- p.64Chapter 3.2 --- Morphology and GFAP Expression in Cytokine-treated C6 Glioma Cells --- p.64Chapter 3.2.1 --- Effects of Cytokines on the Morphology of C6 Cells --- p.64Chapter 3.2.2 --- Effects of Cytokines on GFAP Expression in C6 Glioma Cells --- p.66Chapter 3.3 --- The Signalling Pathway of Cytokine-induced C6 Cell Proliferation --- p.69Chapter 3.3.1 --- The Involvement of Protein Kinase C in Cytokine-induced C6Cell Proliferation --- p.71Chapter 3.3.2 --- The Involvement of Tyrosine Kinase in the Cytokine- induced C6 Cell Proliferation --- p.81Chapter 3.3.3 --- The Involvement of Calcium Ions in Cytokine-induced C6 Cell Proliferation --- p.87Chapter 3.3.4 --- The Involvement of Cyclic Nucleotides in Cytokine- induced C6 Cell Proliferation --- p.92Chapter 3.3.5 --- The Involvement of Nitric Oxide in Cytokine-induced C6 Cell proliferation --- p.94Chapter 3.3.6 --- The Involvement of P-Adrenergic Receptor in Cytokine- induced C6 Cell Proliferation --- p.101Chapter Chapter 4: --- Discussion and Conclusions --- p.104References --- p.11

Regulation of cytosolic Phospholipase A2 activity plays a central role in cell responses

Phospholipases A2 are enzymes that hydrolyse fatty acids from the sn-2 position of phospholipids, resulting in the release of free fatty acids and lysophospholipids. The sn-2 position of phospholipids in mammalian cells is enriched with arachidonic acid, which is a substrate for cyclooxygenases, lipoxygenases and cytochrome p450s, giving PLA2s an important role in the control of the synthesis of prostaglandins, leukotrienes and other eicosanoids. Arachidonic acid and its metabolites, the eicosanoids, have been implicated in a number of physiological and pathophysiological processes and is preferentially released by cytosolic phospholipase A2 (cPLA2), implicating that cPLA2 activity has to be tightly regulated. The aim of this thesis was to gain more insight in the regulation of cPLA2 in mitogen- and oxidative stress-induced cells, as well as in continuously cycling cells. Furthermore, the possible role of cPLA2 and the downstream arachidonic acid metabolising enzymes, cyclooxygenases and lipoxygenases, in cell cycle progression was investigated. The studies described in chapters 2, and 3 show that cPLA2 is activated through different signal transduction pathways depending on the stimulus in the extracellular environment. In response to epidermal growth factor (EGF), cPLA2 is predominantly activated through PKC-MEK-p42/44MAPK, while serum-induced cPLA2 activity is mainly mediated via the Raf-MEK-p42/44MAPK pathway. In contrast, direct activation of PKC by phorbol ester (PdBu) did not result in increased cPLA2 activity, while p42/44MAPK was activated via Raf-MEK and through MEK. Activation of cPLA2 by the oxidant hydrogen peroxide (H2O2) is partly mediated via Raf-MEK-p42/44MAPK and partly through a phosphorylation-independent mechanism involving peroxidised phospholipids. These results suggest that activation of cPLA2 is not only governed by post-translational modifications but, more importantly, by localisation of the signal transduction components at a certain time that determines whether cPLA2, at which place and what time cPLA2 will be activated. The cellular localisation of signal transduction components determines whether cPLA2 will be activated. However, understanding the function of cPLA2 in cells requires also knowledge of the activation of cPLA2 in a temporal manner. Therefore, the activity and function of cPLA2 was investigated during the ongoing cell cycle as described in chapters 4 and 5. cPLA2 activity was high in mitosis, decreasing rapidly in early G1. A small increase was observed in mid/late G1, followed by a strong increase at the G1/S transition. These changes in cPLA2 activity were not due to differences in cPLA2 protein expression, but due to p42/44MAPK mediated phosphorylation of the enzyme. Inhibition of cPLA2 activity in early G1 phase using ATK, an inhibitor for cPLA2, resulted in a marked reduction in DNA synthesis. Furthermore, inhibition of cyclooxygenases at different time points after mitosis did not have any effect on cell cycle progression from G1 to S phase, whereas inhibition of lipoxygenases resulted in G1 phase arrest. Moreover, lipoxygenases are pivotal for S phase progression, since no DNA synthesis occurred when lipoxygenases were inhibited. Thus it is important to understand the mechanism and function of cPLA2 regulation for the development and therapeutic use of drugs