15-hydroxy-5-8-11-13-eicosatetraenoic-acid and stearic-acid

The ubiquitous hydroxylated fatty acids derived from arachidonic acid (HETEs) or linoleic acid (HODEs) exhibit diverse biological effects including chemotaxis, cell proliferation, and modulation of several enzymatic pathways, including the 5-lipoxygenase leading to the inflammatory leukotrienes. It was observed that 12(S)- and 15(S)-HETE and 13(S)-HODE (12- and 15-lipoxygenase-derived metabolites, respectively) inhibited the 5-lipoxygenase present in rat basophilic leukemia (RBL-1) cell homogenates whereas the 15(R) chiral enantiomer and the nonhydroxylated linoleic, oleic, and stearic acids were either less potent or ineffective. In examining the mechanism of this inhibition, the relative effectiveness of several fatty acids in displacing [3H]15-HETE bound to cytosol preparations were compared and the results indicated that these (S) hydroxy fatty acids and 5(S)-HETE were significantly more potent than either the 15(R) enantiomer, 15(S)-HETE methyl ester, arachidonic acid, or prostaglandin F2alpha. In order to identify the protein(s) that specifically binds HETEs, 15(S)-HETE biotin hydrazide was used as a probe to detect any HETE-protein complexes as this compound both inhibited the 5-lipoxygenase and interfered with the binding of [3H]15-HETE to cytosol preparations. SDS-PAGE analysis and chemiluminescent detection revealed that the major cytosolic proteins that bound this biotinylated probe had molecular masses of 43 and 51 kD. Fatty acid competition experiments indicated that the order of effectiveness in displacing this probe from these proteins was 13(S)-HODE > 5(S)-HETE approximately equal to 15(S)-HETE > > stearic acid approximately equal to arachidonic acid approximately equal to 15(R)-HETE. Amino acid sequence analysis showed that the 43 kD protein was actin. These findings suggest the possibility that actin may play a major role in the biological effects of monohydroxylated metabolites derived from cellular 5-, 12-, and 15-lipoxygenases.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Actins; Amino Acid Sequence; Animals; Arachidonate 15-Lipoxygenase; Biotinylation; Carrier Proteins; Cytosol; Dinoprost; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia, Basophilic, Acute; Ligands; Linoleic Acid; Linoleic Acids; Molecular Sequence Data; Myelin P2 Protein; Neoplasm Proteins; Nerve Tissue Proteins; Oleic Acid; Rats; Stearic Acids; Stereoisomerism; Structure-Activity Relationship; Tumor Cells, Cultured

2,3,5-Trimethyl-6-(3-pyridylmethyl) 1,4-benzoquinone (CV-6504), an inhibitor of 5-lipoxygenase, effectively suppressed growth of the MAC16 tumour in vivo and prevented the accompanying cachexia, when administered daily at a dose of 10 mg kg(-1). There was a reduction in the tumour concentration of linoleic (LA), arachidonic (AA), oleic, stearic and palmitic acid. In order to elucidate the mechanism of the anti-tumour action, the effect of CV-6504 on the metabolism of AA through the 5-, 12- and 15-lipoxygenase pathways has been determined in cell lines sensitive (MAC16, MAC13, MAC26 and Caco-2) and resistant (A549 and DU-145) to CV-6504. Incubation of all cell lines with [3H]AA led to the appearance of [3H]5-, 12- and 15-HETE. Preincubation of MAC16, MAC13, MAC26 and Caco-2 with 10 microM CV-6504 inhibited the conversion of AA to 5-, 12- and 15-HETE, while in A549 and DU-145 cells there was no effect on metabolism through any lipoxygenase pathway. Two other cell lines, MDA-MB-231 and PC-3, sensitive to growth inhibition by CV-6504, are known to require LA for growth, while DU-145, which was insensitive to growth inhibition by CV-6504, showed no growth response to LA. These results suggest that some tumours are dependent on lipoxygenase metabolites of LA and AA for their continual growth, and interference with this pathway produces a specific growth inhibition.

Topics: alpha-Linolenic Acid; Animals; Antineoplastic Agents; Arachidonic Acids; Benzoquinones; Cachexia; Drug Screening Assays, Antitumor; Female; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors; Mice; Mice, Inbred Strains; Oleic Acid; Palmitic Acid; Stearic Acids; Tumor Cells, Cultured