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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要： 背景：我们鉴定了一种来自玉米芯铺垫材料的内分泌干扰物，它可以干扰大鼠的雄性和雌性性行为和卵巢周期，并刺激雌激素受体(ER)阳性和ER阴性的乳腺癌细胞增殖。这些试剂被鉴定为四氢呋喃二醇(9，12-氧基-10，13-二羟基十八酸和10，13-氧基，9，12-二羟基-十八酸)的异构体混合物。合成的四氢呋喃二醇在0.5-1ppm的口服浓度下抑制大鼠的性行为，并在体外刺激MCF-7人乳腺癌细胞的增殖。 目的：由于四氢呋喃二醇来源于脂氧合酶和环氧合酶途径，我们推测这些化合物可能通过调节参与亚油酸代谢的磷脂酶A2(PLA2)、脂氧合酶(LOX-5和LOX-12)、环氧合酶(COX-1和COX-2)以及紧密耦合的酶(包括芳香酶)来调节细胞增殖。 方法：用磷脂酶A2(PLA2)、脂氧合酶(LOX-5和LOX-12)、环氧合酶(COX-1、COX-2、吲哚美辛)和芳香酶(福美斯坦)抑制剂处理MCF-7人乳腺癌细胞。研究了这些酶抑制剂对四氢呋喃二醇或雌二醇(E2)诱导的细胞增殖的影响。四氢呋喃二醇对这些酶的表达(RNA和蛋白质)的调节也通过实时定量聚合酶链式反应(QPCR)和蛋白质印迹分析进行了评估。 结果：酶抑制和基因表达(RNA和蛋白质)研究证实PLA2、LOX-5、LOX-12和COX-2以及芳香化酶可能是四氢呋喃二醇调节MCF-7细胞的可能部位。四氢呋喃二醇对COX-1无明显影响。 讨论：四氢呋喃二醇对MCF-7细胞增殖的刺激是通过影响PLA2、COX-2、LOX-5和LOX-12的表达和/或活性来实现的。由于这些酶的产物，包括前列腺素、羟基二十碳四烯酸(HETE‘s)和羟基十八烯酸(HODE’s)，是正常和恶性细胞中广泛存在的有丝分裂原，这些化合物很可能参与了四氢呋喃二醇在乳腺癌细胞中的作用机制。虽然福美斯坦抑制研究表明芳香酶活性可能受到四氢呋喃二醇的调节，但这一点并未得到基因表达研究的证实。 关键词：四氢呋喃二醇、PLA2、COX-2、LOX-5、LOX-12基因表达，乳腺癌细胞增殖。

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.