THF-DIOL STIMULATION OF PLA2, LOX AND COX GENE EXPRESSION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Abstract: Background: We characterized an endocrine disrupter from ground corncob bedding material that interferes with male and female sexual behavior and ovarian cyclicity in rats and stimulates estrogen receptor (ER)-positive and ER-negative breast cancer cell proliferation. The agents were identified as an isomeric mixture of THF-diols (9, 12-oxy-10, 13-dihydroxyoctadecanoic acid and 10, 13-oxy, 9, 12-dihydroxy-octadecanoic acid). Synthetic THF-diols inhibited rat male and female sexual behavior at oral concentrations of 0.5 to 1 ppm, and stimulated MCF-7 human breast cancer cell proliferation in vitro. Objectives: Since THF-diols are derived from lipoxygenase and cyclooxygenase pathways, we suspected that these compounds may regulate cell proliferation by modulating specific enzymatic sites involved in linoleic acid metabolism including phospholipase A2 (PLA2), lipoxygenases (LOX-5 and LOX-12), cyclooxygenases (COX-1 and COX-2) and closely coupled enzymes including aromatase. Methods: MCF-7 human breast cancer cells were treated with inhibitors for phospholipase A2 (PLA2; Quinacrine), lipoxygenases (LOX-5 and LOX-12; Baicalein, Rev 5091, NDGA), cyclooxygenases (COX-1, COX-2, Indomethacin) and aromatase (Formestane). The effects of these enzyme inhibitors on cell proliferation in response to THF-diols or estradiol (E2) were assessed. THF-diol modulation of the expression (RNA and protein) of these enzymes was also evaluated by real time PCR (QPCR) and Western blot analyses. Results: The enzyme inhibition and gene expression (RNA and protein) studies identified PLA2, LOX-5, LOX-12 and COX-2 and perhaps aromatase as likely sites of THF-diol regulation in MCF-7 cells. COX-1 was not affected by THF-diol treatment. Discussion: THF-diol stimulation of MCF-7 cell proliferation is mediated through effects on the expression and/or activities of PLA2, COX-2, LOX-5 and LOX-12. As the products of these enzymes, including prostaglandins, hydroxyeicosatetraenoic acids (HETE's) and hydroxyoctadecenoic acids (HODE's), are well-established mitogens in normal and malignant cells, it is likely that these compounds are involved in the mechanism of action of THF-diols in breast cancer cells. Although the Formestane inhibition studies suggested that aromatase activity may be modulated by THF-diols, this was not confirmed by the gene expression studies. Key Words: THF-diols, PLA2, COX-2, LOX-5, LOX-12 gene expression, breast cancer cell proliferation.