15-hydroxy-5-8-11-13-eicosatetraenoic-acid and Breast-Neoplasms

Lipoxygenase (LOX) are enzymes implicated in a broad range of inflammatory diseases, cancer, asthma and atherosclerosis. These diverse biological properties lead to the interesting target for the inhibition of this metabolic pathway of LOX. The drugs available in the market against LOX reported to have various side effects. To develop potent and selective therapeutic agents against LOX, it is essential to have the knowledge of its active site. Due to the lack of structural data of human LOX, researchers are using soybean LOX (sLOX) because of their availability and similarities in the active site structure. Based on the crystal structure of sLOX-3 and its complex with known inhibitors, we have designed a tripeptide, FWY which strongly inhibits sLOX-3 activity. The inhibition by peptide has been tested with purified sLOX-3 and with LOX present in blood serum of breast cancer patients in the presence of substrate linoleic acid and arachidonic acid respectively. The dissociation constant (K(D)) of the peptide with sLOX-3 as determined by Surface Plasmon Resonance (SPR) was 3.59x10(-9) M. The kinetic constant (K(i)) and IC(50), as determined biochemical methods were 7.41x10(-8) M and 0.15x10(-6) M respectively.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Breast Neoplasms; Drug Design; Drug Screening Assays, Antitumor; Enzyme Activation; Female; Glycine max; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Lipoxygenase; Lipoxygenase Inhibitors; Protein Binding; Surface Plasmon Resonance

Conjugated linoleic acid (CLA) consists of a group of linoleic acid geometric isomers that have been shown to reduce tumor growth and metastasis in animal models of breast, prostate and colon cancer. To delineate a possible mechanism of action for CLA, we have recently shown that the 5-lipoxygenase product, 5-hydroxyeicosatetraenoic acid (5-HETE), could play a role in CLA alteration of mammary tumorigenesis. In this study, we determined how CLA could modulate 5-lipoxygenase activity. The t10, c12-CLA isomer reduced production of 5-HETE but not 12- and 15-HETE in MDA-MB-231 human breast tumor cells. That isomer and the c9, t11-CLA isomer decreased 5-HETE production by competition with the lipoxygenase substrate, arachidonic acid (AA). Interestingly, t10, c12-CLA reduced the expression of five-lipoxygenase activating protein (FLAP) but not the 5-lipoxygenase enzyme. Over-expression of FLAP abrogated t10, c12-CLA-reduced viability of MDA-MB-231 cells. These data suggest that the reduction of 5-HETE by t10, c12-CLA was due to competition with AA and the reduction of FLAP expression.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Isomerism; Linoleic Acids; Linoleic Acids, Conjugated; Membrane Proteins; Transfection

The relationship between 15(S)-HETE and 13(S)-HODE from different human tumor cells exposed to n-6 and n-3 essential fatty acids (EFAs) and E-cadherin expression was studied. Colon cancer cells (HRT-18) exposed to gamma linoleic acid (18:3n-6, GLA) and eicosapentaenoic (20:5n-3, EPA) (50microM) showed an increased expression of E-cadherin. Breast cancer (MCF-7) exposed to EPA showed an increment whereas GLA had no effect on E-cadherin expression. No expression of E-cadherin was observed for urothelial cancer (T-24) after GLA or EPA treatment. Significant levels of 15(S)-HETE and 13(S)-HODE were detected after GLA or EPA treatment for all tumor lines. E-cadherin expression was inversely proportional to the 13(S)-HODE:15(S)-HETE ratio when cells were pretreated with GLA or EPA. Nevertheless, the liberation of these metabolites seems to be independent of the E-cadherin expression. The increase in the13(S)-HODE:15(S)-HETE correlates to a decrease in the expression of E-cadherin. Both factors may play a role in metastasis development.

Topics: Arachidonic Acid; Breast Neoplasms; Cadherins; Cell Differentiation; Colonic Neoplasms; Female; Humans; Hydroxyeicosatetraenoic Acids; Immunohistochemistry; Linoleic Acid; Linoleic Acids; Neoplasm Metastasis; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Urothelium

Diets rich in linoleic acid (LA) stimulate the metastasis of MDA-MB-435 human breast cancer cells from the mammary fat pads of nude mice. This omega-6 fatty acid is metabolized to various cyclo-oxygenase and lipoxygenase products, several of which have been previously associated with tumor cell invasion and metastasis. We now report that MDA-MB-435 cells secreted increased levels of prostaglandin E2 (PGE2), and 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE when cultured in the presence of 2.7 microM (0.75 micrograms/ml) LA; 5-HETE secretion was unchanged. The 12-lipoxygenase inhibitor esculetin (20 microM) completely blocked the LA-stimulated 12-HETE secretion. Linoleic acid also increased MDA-MB-435 cell invasion in an in vitro assay; this stimulation was abolished by 20 microM esculetin, but was unaffected by piroxicam, a selective cyclooxygenase inhibitor. The effect of LA on invasion was replicated by 0.1 microM 12-HETE, but not by 5-HETE or PGE2; 15-HETE was stimulatory only at a concentration of 1.0 microM. Zymographic and Northern blot analyses showed that these events are accompanied by the induction of 92 kDa isoform type IV collagenase (metalloproteinase-9) enzymic activity and mRNA expression by exogenous LA and 12-HETE, and their suppression by the 12-lipoxygenase inhibitor. These results suggest that the effects of dietary LA on breast cancer cell metastasis in the nude mouse model are due, at least in part, to enhanced 12-HETE biosynthesis, with an associated increase in proteolytic enzyme activity and tumor cell invasiveness.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Breast Neoplasms; Collagenases; Eicosanoids; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Lipoxygenase Inhibitors; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Piroxicam; RNA, Messenger; Tumor Cells, Cultured; Umbelliferones

There is evidence suggesting a role of eicosanoids in the growth of certain tumors. In this study, tissue samples were collected from basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) of the skin. Both BCCs and SCCs contained more prostaglandin E2 and F2 alpha (PGE2 and PGF2 alpha) than normal epidermis. In vitro incubation of tumor samples with arachidonic acid also resulted in PGE2 and PGF2 alpha formation. Basal cell carcinomas exhibiting a histologically aggressive growth pattern contained higher levels of prostaglandins than those with a nonaggressive growth pattern, both in vivo and after in vitro incubation. Lipoxygenase products (12- and 15-hydroxyeicosatetraenoic acid) were present in smaller amounts than cyclo-oxygenase products (PGE2 and PGF2 alpha) in vivo. Compared with normal epidermis, SCCs and, particularly, BCCs produced smaller amounts of 12-hydroxyeicosatetraenoic acid during in vitro incubation with arachidonic acid. The levels of lipoxygenase products were not related to the tumor growth pattern. These results indicate that excessive prostaglandin levels in BCCs may be associated with an aggressive growth pattern.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Breast Neoplasms; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Chromatography, High Pressure Liquid; Dinoprost; Dinoprostone; Head and Neck Neoplasms; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Mice; Prostaglandins; Prostaglandins E; Prostaglandins F; Rabbits; Radioimmunoassay; Skin Neoplasms