Arachidonic Acid Cytochrome P450 Epoxygenase 2J2 Promotes Human Tumor Metastasis

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.