leukotriene-b4 and Brain-Neoplasms

Exogenous application of beta-amyloid (Abeta(25-35), a fragment of Abeta(1-42)) significantly elevated levels of reactive oxygen species (ROS) in C6 astroglioma cells, as measured by confocal microscopic analysis of H(2)O(2)-sensitive 2',7'-dichlorofluorescin fluorescence. Subsequent characterization of the signalling pathway revealed that expression of RacN17, a dominant-negative Rac1 mutant, completely blocked Abeta(25-35)-induced generation of ROS, which is indicative of the crucial role played by Rac GTPase in this process. To better understand the downstream mediators affected by Rac, we assessed the degree to which inhibition of cytosolic phospholipase A(2) (cPLA(2)) and 5-lipoxygenase (5-LO) contributed to the response and found that inhibition of either enzyme completely blocked Abeta(25-35)-induced ROS generation, indicating its dependence on arachidonic acid synthesis and metabolism to leukotrienes (e.g. leukotriene B(4)). Consistent with those findings, Abeta(25-35) Rac-dependently stimulated translocation of 5-LO to the nuclear envelope and increased intracellular levels of leukotriene B(4), while exogenous application of leukotriene B(4) increased intracellular H(2)O(2) via BLT, its cell-surface receptor. In addition to the aforementioned downstream mediators, inhibition of phosphoinositide 3-kinase (PI 3-kinase), an enzyme situated upstream of Rac, also completely blocked Abeta(25-35)-induced H(2)O(2) generation. Our findings thus demonstrate that PI 3-kinase, Rac, cPLA(2) and 5-LO are all essential components of the beta-amyloid signaling cascade leading to generation of ROS.

Topics: Amyloid beta-Peptides; Arachidonate 5-Lipoxygenase; Astrocytoma; Blotting, Western; Brain Neoplasms; Cytosol; Humans; Hydrogen Peroxide; Leukotriene B4; NADPH Oxidases; Phosphatidylinositol 3-Kinases; Phospholipases A; Plasmids; rac1 GTP-Binding Protein; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Subcellular Fractions; Time Factors; Transfection; Tumor Cells, Cultured

The role of intracellular Ca2+ in the regulation of tumor cell proliferation by products of arachidonic acid (AA) metabolism was investigated using U-373 MG human astrocytoma cells. Treatment with nordihydroguaiaretic acid (NDGA), a lipoxygenase (LOX) inhibitor, or caffeic acid (CA), a specific 5-LOX inhibitor, suppressed proliferation of the tumor cells in a dose-dependent manner. However, indomethacin (Indo), a cyclooxygenase (COX) inhibitor, did not significantly alter proliferation of the tumor cells. At anti-proliferative concentrations, NDGA and CA significantly inhibited intracellular Ca2+ release induced by carbachol, a known intracellular Ca2+ agonist in the tumor cells. Exogenous administration of leukotriene B4 (LTB4), an AA metabolite of LOX pathway, enhanced proliferation of the tumor cells in a concentration-dependent fashion. In addition, LTB4 induced intracellular Ca2+ release. Intracellular Ca2+ inhibitors, such as an intracellular Ca2+ chelator (BAPTA) and intracellular Ca(2+)-release inhibitors (dantrolene and TMB-8), significantly blocked the LTB4-induced enhancement of cell proliferation and intracellular Ca2+ release. These results suggest that LOX activity may be critical for cell proliferation of the human astrocytoma cells and that intracellular Ca2+ may play a major role in the mechanism of action of LOX.

Topics: Arachidonic Acid; Astrocytoma; Brain Neoplasms; Calcium; Calcium Channel Blockers; Cell Division; Humans; Ion Transport; Leukotriene B4; Lipoxygenase; Tumor Cells, Cultured