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

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 摘要： 背景资料：我们的特点是从地面玉米芯垫料的内分泌干扰物，干扰男性和女性的性行为和卵巢周期性大鼠和刺激雌激素受体（ER）阳性和ER阴性乳腺癌细胞增殖。该试剂被鉴定为THF-二醇（9，12-氧-10，13-二羟基十八烷酸和10，13-氧，9，12-二羟基十八烷酸）的异构体混合物。合成的THF-二醇在0.5至1 ppm的口服浓度下抑制大鼠雄性和雌性的性行为，并在体外刺激MCF-7人乳腺癌细胞增殖。 目的：由于THF-二醇来源于脂氧合酶和环氧合酶途径，我们怀疑这些化合物可能通过调节亚油酸代谢中涉及的特定酶位点来调节细胞增殖，所述酶位点包括磷脂酶A2（PLA 2）、脂氧合酶（LOX-5和LOX-12）、环氧合酶（考克斯-1和考克斯-2）和紧密偶联的酶（包括芳香酶）。 研究方法：用磷脂酶A2（PLA 2;奎纳克林）、脂氧合酶（LOX-5和LOX-12;黄芩素，Rev 5091，NDGA）、环氧合酶（考克斯-1，考克斯-2，吲哚美辛）和芳香酶（福美坦）的抑制剂处理MCF-7人乳腺癌细胞。评估这些酶抑制剂对响应于THF-二醇或雌二醇（E2）的细胞增殖的影响。 还通过真实的时间PCR（QPCR）和蛋白质印迹分析来评估这些酶的表达（RNA和蛋白质）的THF-二醇调节。 结果如下：酶抑制和基因表达（RNA和蛋白质）研究确定PLA 2、LOX-5、LOX-12和考克斯-2以及可能的芳香酶是MCF-7细胞中THF-二醇调节的可能位点。考克斯-1不受THF-二醇处理的影响。 讨论内容：THF-二醇对MCF-7细胞增殖的刺激是通过对PLA 2、考克斯-2、LOX-5和LOX-12的表达和/或活性的影响来介导的。由于这些酶的产物，包括胡萝卜素、羟基二十碳四烯酸（HETE）和羟基十八碳烯酸（HODE），是正常和恶性细胞中公认的促有丝分裂剂，因此这些化合物可能参与乳腺癌细胞中THF-二醇的作用机制。 尽管福美坦抑制研究表明芳香酶活性可能受THF-二醇调节，但这未被基因表达研究证实。 关键词：四氢呋喃二醇，磷脂酶A2，考克斯-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.