【作者】 宁耀贵；

【导师】 汪道文；

【作者基本信息】 华中科技大学 ， 内科学， 2006， 硕士

【摘要】 目的与意义花生四烯酸(arachidonic acid, AA)是生物体内最丰富的物质之一,是体内多种重要活性物质的来源,主要是以酯化的形式结合在细胞膜内侧的脂肪酸上,在脂解激素刺激时由磷脂酶A2水解释放至细胞浆内。对AA的研究已有多年,最为人们熟知的是环氧化酶和脂质氧化酶代谢途径,近年研究发现AA还可通过细胞色素P450途径代谢(即AA代谢的第三条途径),包括花生四烯酸细胞色素P450表氧化酶(AA cytochrome P450 epoxygenase, CYP)途径和ω-羟化酶途径,其中AA经表氧化酶代谢产生四种EETs (5,6-, 8,9-, 11,12-和14,15-EET)。已经发现在哺乳动物有14个家族和26个亚族,而在人类仅发现有CYP2C和CYP2J表达。CYP在体内广泛分布,尤其在肝脏中表达最丰富,主要存在于内质网和线粒体,在血管中亦存在表达;目前已发现并克隆2J家族的9个基因,其中在人类仅发现了2J2,其在心脏和血管内皮细胞表达最丰富,我们和国外学者已经发现EETs在调节心血管系统稳态中发挥着重要作用,EETs可以通过激活钙离子敏感的钾通道,使平滑肌细胞处于超极化状态而扩张血管调节血压,还参与调节炎症、细胞迁移、细胞凋亡、血小板聚集和保护内皮细胞。我们的研究首次证实表氧化酶基因在不同的人类肿瘤组织中以及肿瘤细胞系中选择性高表达。研究发现转染CYP2J2和外源性EETs通过激活MAPK、PI3K/AKt、及EGFR途径促进肿瘤的恶性增殖;通过上调抗凋亡蛋白Bcl-2和Bcl-xL、下调凋亡蛋白Bax来保护肿瘤细胞凋亡。这些结果表明CYP2J2在人类肿瘤的发生和发展中发挥重要的作用。然而CYP和EETs在人类肿瘤转移中的作用及其机制如何,花生四烯酸表氧化酶抑制剂在肿瘤的防治中有何意义呢?本研究通过对雌激素受体阴性、高转移的乳腺癌细胞(MDA-MB-435s)给予重组腺相关病毒(recombinant adeno-associated virus,rAAV)介导的表氧化酶基因转染,探讨其对肿瘤转移的影响以及相关机制;试验了花生四烯酸表氧化酶抑制剂(17-ODYA)对裸鼠移植瘤的抑瘤效应。方法与结果1.培养MDA-MB-435s细胞,通过重组腺相关病毒介导的正义和反义CYP2J2转染,主要进行以下研究1.1 CYP2J2对MDA-MB-435s细胞增殖的影响:肿瘤是由于致瘤因素的作用,局部组织的细胞失去了对其正常生长的调控,导致异常克隆性增生。正常细胞生长依赖锚泊,有密度依赖抑制或接触抑制,肿瘤细胞则缺乏这种生长限制,甚至可在半固体琼脂中呈悬浮生长,不需要依附固定表面,不受密度限制,可持续分裂堆积生长。琼软脂克隆形成又叫锚泊不依赖生长,通过计数2-4周后软琼脂上的克隆数目来检测细胞的增殖能力,细胞在这种半固态媒体上生长是表型改变的指标之一,也是证明促有丝分裂能力的方法之一。因此我们利用软琼脂克隆形成试验来观察CYP2J2转染后在软琼脂上形成克隆的能力。结果反义2J2转染明显抑制MDA-MB-435s在软琼脂上形成克隆的能力,与GFP组(10.75±2.753)和空白对照组(11.75±2.217)比较,反义2J2转染组(3.25±2.217)形成的克隆数目少,直径小;CYP2J2(19±3.7416)转染则显著促进MDA-MB-435s细胞在软琼脂上形成克隆,且克隆较对照组和转染GFP组明显增大,而且数目多。1.2 CYP2J2对MDA-MB-435s细胞黏附于细胞外基质的影响:肿瘤的浸润过程首先是肿瘤细胞黏附并降解细胞外基质,然后迁移穿过细胞外基质。我们选取细胞外基质主要成分之一纤维粘连蛋白(fibronectin, FN)来研究表氧化酶及其产物对肿瘤细胞黏附的影响。结果表明,与对照组比较,转染反义CYP2J2显著抑制MDA-MB-435s细胞黏附于基质成分黏附细胞数约是对照组的50%,而转染CYP2J2使其过表达表氧化酶则显著促进MDA-MB-435s细胞黏附(P<0.05)。用表氧化酶代谢花生四烯酸产物EETs预处理MDA-MB-435s半小时也显著的促进MDA-MB-435s细胞黏附,而表氧化酶抑制剂能显著抑制MDA-MB-435s细胞的黏附(P<0.01)。1.3 CYP2J2对MDA-MB-435s细胞游走迁移的影响:细胞的游走迁移是肿瘤浸润转移的一个重要步骤。我们利用改良的Boyden趋化小室研究CYP2J2对MDA-MB-435s细胞游走迁移的影响。实验结果显示转染反义CYP2J2的MDA-MB-435s细胞穿过多聚碳酸酯膜的细胞数下降49%,而转染正义CYP2J2的细胞穿过多聚碳酸酯膜的细胞数几乎为对照组的两倍,外源性应用表氧化酶抑制剂(17-ODYA)明显抑制MDA-MB-435s细胞的迁移,而三种EETs显著促进MDA-MB-435s细胞迁移。1.4 CYP2J2对肿瘤转移影响的在体实验研究及其可能机制:为了观察表氧化酶基因2J2是否影响肿瘤的在体转移能力,我们用rAAV-2J2、rAAV-anti2J2和rAAV-GFP感染MDA-MB-435s细胞,3天后接种至雌性裸鼠腋窝皮下,观察肿瘤生长情况。12周后处死裸小鼠,bouin’s solution固定肺组织,观察比较肺部转移瘤结节数目。结果显示CYP2J2转染后的肿瘤细胞在体内生长迅速,肿瘤结节出现的平均时间明显提前,瘤结节体积与对照组比较体积明显增大;转染反义CYP2J2的肿瘤细胞在体生长缓慢,肿瘤结节出现时间延迟,平均瘤结节体积比对照组明显减小,其中有三只在12周末仍然没有长出可以目测的移植瘤,与对照组比较差别有显著性意义(P<0.05)。接着我们观察了肿瘤在肺部的转移情况,结果发现CYP2J2显著促进了肿瘤的肺转移,肺组织表面肉眼能见的瘤结节数目为对照组的2.8倍,反义CYP2J2组则没有观察到明显的肺转移。以上结果显示CYP2J2具有促恶性肿瘤转移的作用。在此基础上,我们研究了CYP2J2和EETs促进肿瘤转移的可能机制,用Western blot方法检测EETs干预和表氧化酶基因转染对转移相关基因表达的影响,同时还用Gelatin Zymography方法检测了CYP2J2对MMPs分泌的影响。结果发现CYP2J2促进MMP-2的分泌;反义CYP2J2则抑制MMP-2的分泌;EETs和CYP2J2明显上调CD44的表达,下调nm23和CD82/KAI的表达,反义CYP2J2或CYP抑制剂则显著下调CD44的表达,上调nm23和CD82/KAI的表达。2.花生四烯酸表氧化酶抑制剂(17-ODYA)对人舌鳞癌裸鼠移植瘤抑瘤效应的实验研究将人舌鳞癌细胞系(Tca 8113)接种至BALB/c(nu/nu)裸鼠,建立人舌鳞癌裸鼠移植瘤模型,设立阴性对照组(N.S)、阳性对照组(5-FU 30mg/kg)、低剂量抑制剂组(0.5mg/只)、高剂量抑制剂组(1mg/只)和联合用药组(0.5mg/只17-ODYA+30mg/kg5-FU),观察肿瘤的生长状态,计算抑瘤率;观察移植瘤的组织病理学及超微结构改变。结果与阴性对照组比较,花生四烯酸表氧化酶抑制剂(17-ODYA)组裸鼠皮下移植瘤体积明显缩小(P<0.01),移植瘤的生长明显受到抑制;高剂量组平均肿瘤体积与低剂量组比较,抑制作用更明显,但两者之间差异无统计学意义;17-ODYA组与阳性对照组相比,高剂量抑制剂组肿瘤平均体积减小不如阳性对照组明显,但两组之间无统计学差异;联合用药组与其他组相比肿瘤明显受到抑制(P<0.01)。瘤结节的重量比较与瘤结节体积结果一致。结论:我们的研究结果显示①CYP2J2及其代谢花生四烯酸的下游产物EETs在促进肿瘤的恶性增殖、促进肿瘤细胞与细胞外基质的黏附以及促进肿瘤细胞游走运动能力等多个环节促进恶性肿瘤转移,同时还可能与促进MMPs的分泌、上调转移相关基因CD44的表达、下调转移抑制基因CD82/KAI和nm23的表达有关。②花生四烯酸细胞色素P450表氧化酶抑制剂(17-ODYA)对人舌鳞癌裸鼠移植瘤有明显的抑瘤效应,但是其作用机制、毒副作用以及与其他临床化疗药物联合的疗效尚需进一步的研究。总之,这些发现为恶性肿瘤的基因治疗提供了新的靶点,为开发新的抗癌药物提供了理论基础。但是恶性肿瘤的发生发展是一个多因素多步骤的过程,CYP作为一个独立的影响因子其作用机制还没有完全阐明,应用于临床尚有局限性,有待于进一步的研究。更多还原

【Abstract】 Arachidonic acid cytochrome P450 (CYP) epoxygenase converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs) and CYP represents a substantial source of arachidonic acid-derived metabolites and reactive species, which have significant effects on cellular function. Release of arachidonic acid from cell membrane by actived phospholidase A2 renders it accessible for metabolism by three pathways: cyclooxygenases, lipoxygenases,and cytochromes P450. Metabolism of free arachidonic acid by cyclooxygenases leads to formation of prostaglandins, prostacylin, and thromboxanes, with important roles in numerous physiological and pathophysiological process such as vasodilation , inflammation, thrombosisl; Metabolism of free arachidonic acid by lipoxygenases leads to the formation of leukotrienes, with important functions as mediators of a variety of inflammatory and allergic reactions. In the past two decades, a third metabolic pathway was found, CYP pathway, which lead to formation of HETEs and four regioisomeric epoxyeicosatrienoic acids (5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET). The cytochrome P450 enzymes comprise a large superfamily of proteins, abbreviated as CYP enzymes, classified in different families (denoted by an Arabic numeral) and subfamilies (indicated by a letter) in accordance with the degree of homology of amino acid sequence in their protein structure. In mammals, 14 families and 26 subfamilies of cytochromes P450 have been identified. Cytochromes P450 can be found in nearly every tissue, being more abundantly expressed in the liver, and are localized in the endoplasmic reticulum and mitochondria (molecular weight 50–60 kDa), in which CYP2J2 prominently existed in heart and endothelial cells of human being. CYP metabolites and reactive species have significant effects on cellular function. EETs have been demonstrated to hyperpolarize and relax vascular smooth muscle cells by activating calcium-sensitive potassium channels. P450-dericed metabolites also can activate intracellular second messenger systems that involved in the regulation of inflammation, cell migration, apoptosis, and platelet aggregation. EETs activate tyrosine kinase, ERK1/2, p38MAPK kinase, and PI3K kinase signaling pathways in endothelial and epithelial cells.Our previously study firstly demonstrated CYP2J2 highly and selectly expressed in human carcinomas and tumor cell lines. Transfection of CYP2J2 and exogenous EETs promoted tumor cells malignant proliferation, but blocking CYP2J2 expression by antisense technique or epoxygenase inhibitor 17-ODYA markedly inhibited that their proliferation. This process involved in phosphorylation of epidermal growth factor receptor and activation downstream PI3K and mitogen-activated protein kinase signaling pathways. CYP2J2 and EETs protect carcinoma cells from apoptosis through regulatory effects on proapoptotic and antiapoptotic protein expression. All of these indicate CYP play important role in the development and progression of human tumor. However, the effects of CYP or EETs on metastasis were far from completely known.In this paper, we explored the effect of CYP or EETs on metastasis potential through infection MDA-MB-435s human breast cancer cells with rAAV-2J2, rAAV-anti2J2 and rAAV-GFP. These human breast cancer cells, which were derived from the parent MDA-MB-435 cell line, were used in this study because they display a highly metastatic phenotype. As well, we tested the antitumor effect of CYP inhibitor 17-ODYA in nude mice. Methods and results:1. Study of CYP2J2 promote human tumor metastasis1.1 Clonogenicity Assay of CYP2J2 and EETs on proliferation of MDA-MB-435s cellsNeoplastic transformation occurs via a series of genetic and epigenetic alterations that yield a cell population that is capable of proliferating independently of both external and internal signals that normally restrain growth. For example, transformed cells show reduced requirements for extracellular growth promoting factors, are not restricted by cell-cell contact, and are often immortal. Anchorage-independent growth is one of the hallmarks of transformation, which is considered the most accurate and stringent in vitro assay for detecting malignant transformation of cells. The soft agar colony formation assay is a common method to monitor anchorage-independent growth, which measures proliferation in a semisolid culture media after 3-4 weeks by manual counting of colonies. Results showed that infection MDA-MB-435s cells with rAAV-2J2 significantly promote colony formation in soft agar and clones were larger compared with control or rAAV-GFP infection. In contrast, infection with rAAV-anti2J2 markedly inhibited colony formation and clones were smaller compared with control or rAAV-GFP infection.1.2 Adhession assay of CYP2J2 or EETs on MDA-MB-435s cellsWhen metastasis, tumor cells first bind and adhere to ECM, then degrade ECM and move into a new sites. We choose fibronectin, a major component of ECM, to test CYP2J2 or EETs on MDA-MB-435s cells adhesion potential. Results indicate that transfection with CYP2J2 or exogenous EETs significantly promote MDA-MB-435s cells adhesion to fibronectin. Addition of epoxygenase inhibitor, 17-ODYA, blocking expression of CYP2J2 by rAAV-anti2J2 efficiently inhibited adhesion potential of MDA-MB-435s cells.1.3 Motility Assay of CYP2J2 or EETs on MDA-MB-435s cellsMotility was examined because it is an important component of the invasion process. To determine whether the invasive activity of CYP was attributable to their effect on cell motility, cellular chemotaxis of MDA-MB-435s cells transfected with epoxygenase gene or treated with EETs,17-ODYA toward FCM was tested using Boyden chambers that were prepared with uncoated filters (no Matrigel). Migration cells in the bottom chamber was measured. We demonstrate that both overexpression of CYP2J2 and addition of EETs significantly promoted transwell migration of MDA-MB-435s cells (3 to 5.5 folds of controls); in contrast, epoxygenase inhibitor 17-ODYA and anti-CYP2J2 transfection markedly inhibited cell migration.1.4 Assay of tumor growth and metastasis in animal modelMDA-MB-435s cells were collected three days after infection with rAAV2J2, rAAV-GFP and rAAV-anti2J2, then were injected through a 22-gauge needle mammary fat pad of nude mice. All cells were injected in a volume of 100μl at a concentration of 2×106 viable cells. Tumors were measured every week. Animals were sacrificed 12 weeks, and tumors were excised and weight. The lung were removed ,rinsed in PBS, and placed in Bouin’s solution for 24 hours before counting the number of metastasis sites.In vivo study showed that forced CYP2J2 overexpression promoted growth of tumors and metastasis to lungs by increasing numbers of metastases in lungs, but antisense CYP2J2 transfection inhibited the growth and metastasis. We further explored the mechanism of CYP promote tumor metastasis by immunoblot and Gelatin zymography analysis. We found that CYP2J2 transfection markedly upregulation MMP-9 expression and promote MMP-2 secretion, but rAAV-anti2J2 infection reduced MMP-9 expression and MMP-2 secretion. We probed some metastasis associated molecules by western blot and found that EETs treatment and CYP2J2 overexpression significantly down-regulated expression level of anticancer genes nm-23 and CD82/KAI-1, but P450 inhibitor 17-ODYA treatment and rAAV-anti2J2 infection up-related their expression. On the other hand, EETs treatment and transfection of epoxygenases increase expression level of cancer metastasis enhancing gene CD44, in contrast P450 inhibitor 17-ODYA incubation and rAAV-anti2J2 infection reduced CD44 protein expression level. In the xenograft tumor tissues, we found same pattern of rAAV-2J2 effects on expression of these genes. These results indicate both EETs treatment and P450 epoxygenase gene transfection markedly up-regulate the expression of carcinoma metastasis promoting genes, but down-regulate the expression of cancer metastasis inhibiting genes we have probed.2. Study on Antitumor Effect of 17-ODYA in Treatment of Transplanted Human Squamous Cell Carcinoma of Tongue in Nude MiceTca8113 cells were inoculated subcutaneously in the right flank of BALB/c nude mice. The mice were randomly divided into five groups and received treatment every other day while primary tumor reaching suitable size.①1 7-ODYA 05mg/mouse,②1 7-ODYA 1mg/mouse,③5 -FU 30mg/kg,④17-ODYA 0.5mg/mouse+5-FU 30mg/kg,⑤1 7-ODYA vehicle (3% DMSO plus saline).the antitumor effects of 17-Octadecynoic acid was observed. Results: It showed that subcutaneous tumor growth was significantly inhibited in mice treated with 17-Octadecynoic acid compared with that in controls (P<0.01). The tumor volume and weight in nude mice received 1.0mg/mouse 17-Octadecynoic acid therapy decreased compared with that in nude mice treated with 0.5mg/mouse 17-Octadecynoic acid. Conclusion:It indicated that 17-Octadecynoic acid effectively inhibited growth of human squamous carcinoma of tongue Tca8113 in nude mice.Taken together, our findings suggest that EETs may play important roles in promoting metastasis of human cancers and their mechanisms may be involved in promotion of tumor cells proliferation, adhesion, migration, down-regulation the expression of metastatic suppressor genes and up-regulation of metastatic enhancing genes. Epoxygenase inhibitor 17-Octadecynoic acid, may effectively inhibited growth of human tumor in vivo. These findings together with previous data suggest that CYP epoxygenases are carcinogenic gene and also implicate a novel therapeutic strategy of human carcinomas, through blocking or inhibiting EETs or P450 epoxygenases.更多还原