Inhibition of tumour growth by lipoxygenase inhibitors.

The potential involvement of lipoxygenase metabolites in the tumour growth stimulatory activity of arachidonic and linoleic acid has been studied using the 5-lipoxygenase inhibitors, BWA4C, BWB70C and Zileuton. In vitro the former two agents were relatively potent inhibitors of growth of murine adenocarcinomas (MACs) with IC50 values < 10 microM, whereas Zileuton was less effective. In vivo studies showed BWA4C to be an effective inhibitor of the growth of both the MAC26 and MAC16 tumours at dose levels between 5 and 25 mg kg-1 (b.d.). The growth rate of the MAC26 tumour was also decreased by BWB70C at 25 mg kg-1, whereas lower doses were either ineffective or stimulated tumour growth. This differential effect of the 5-lipoxygenases inhibitors on tumour growth may arise from effects on the 12- and 15-lipoxygenase pathways. To quantify the effect cells were labelled with [3H]arachidonic acid and the biosynthesis of 5-, 12- and 15-hydroxyeicosatetraenoic acid (HETE) was analysed by high-performance liquid chromatography. All three agents caused a decrease in 5-HETE production, although the effect was less pronounced with Zileuton. In MAC26 cells both BWA4C and BWB70C caused a decrease in 12-HETE formation whereas Zileuton had no effect on the other lipoxygenase pathways. The inhibitory effect of these agents on cell growth may result from an imbalance of metabolism of arachidonic acid between the 5-, 12- and 15-lipoxygenase pathways.     

Inhibitory effects of curcumin on in vitro lipoxygenase and cyclooxygenase activities in mouse epidermis

Topical application of curcumin, the yellow pigment in turmeric and curry, strongly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase activity, DNA synthesis, and tumor promotion in mouse skin (Huang et al., Cancer Res., 48: 5941-5946, 1988). Chlorogenic acid, caffeic acid, and ferulic acid (structurally related dietary compounds) were considerably less active. In the present study, topical application of curcumin markedly inhibited TPA- and arachidonic acid-induced epidermal inflammation (ear edema) in mice, but chlorogenic acid, caffeic acid, and ferulic acid were only weakly active or inactive. The in vitro addition of 3, 10, 30, or 100 microM curcumin to cytosol from homogenates of mouse epidermis inhibited the metabolism of arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE) by 40, 60, 66, or 83%, respectively, and the metabolism of arachidonic acid to 8-HETE was inhibited by 40, 51, 77, or 85%, respectively [IC50 (concentration needed for 50% inhibition) = 5-10 microM]. Chlorogenic acid, caffeic acid, or ferulic acid (100 microM) inhibited the metabolism of arachidonic acid to 5-HETE by 36, 10, or 16%, respectively, and these hydroxylated cinnamic acid derivatives inhibited the metabolism of arachidonic acid to 8-HETE by 37, 20, or 10%, respectively (IC50 greater than 100 microM). The metabolism of arachidonic acid to prostaglandin E2, prostaglandin F2 alpha, and prostaglandin D2 by epidermal microsomes was inhibited approximately 50% by the in vitro addition of 5-10 microM curcumin. Chlorogenic acid, caffeic acid, and ferulic acid (100 microM) were inactive. In vitro rat brain protein kinase C activity was not affected by 50-200 microM curcumin, chlorogenic acid, caffeic acid, or ferulic acid. The inhibitory effects of curcumin, chlorogenic acid, caffeic acid, and ferulic acid on TPA-induced tumor promotion in mouse epidermis parallel their inhibitory effects on TPA-induced epidermal inflammation and epidermal lipoxygenase and cyclooxygenase activities.     

Biosynthesis of prostaglandins and hydroxy fatty acids in primary squamous carcinomas of head and neck in humans

The metabolism of [14C]arachidonic acid into cyclooxygenase and lipoxygenase products by homogenates of primary squamous carcinomas of head and neck in 12 patients was studied in vitro. The lipoxygenase pathway was predominant in all samples. The major metabolites were 12-hydroxy-5,8,11-14-eicosatetraenoic acid, (12-HETE) and 15-HETE. 5-HETE, 5,12-diHETE, 8-HETE and 9-HETE were also detected. The cyclooxygenase products detected were in the following order: PGE2 greater than PGF2 alpha greater than TxB2 greater than 15-keto-PGE2 greater than 6-keto-PGF1 alpha greater than PGD2. Literature review of the biological activities of these oxygenated metabolites of arachidonic acid suggest important modulatory roles in the pathophysiology of head and neck cancer.     

Bidirectional control of membrane expression and/or activation of the tumor cell IRGpIIb/IIIa receptor and tumor cell adhesion by lipoxygenase products of arachidonic acid and linoleic acid

Lewis lung carcinoma cells express a plasma membrane receptor (i.e., IRGpIIb/IIIa) which is immunologically and functionally related to the platelet aggregation receptor complex (i.e., GpIIb/IIIa). Both fluorescence microscopy and flow cytometric analysis reveal that surface expression and/or activation of this tumor cell receptor is enhanced by a phorbol ester [i.e., 12-O-tetradecanoylphorbol-13-acetate (TPA)] and a lipoxygenase metabolite of arachidonic acid; 12-hydroxyeicosatetraenoic acid (i.e., 12-HETE). TPA-enhanced expression appears to be mediated by a lipoxygenase metabolite, as this effect can be reversed by lipoxygenase inhibitors but not by cyclooxygenase inhibitors. In parallel with these results both TPA and 12(S)-HETE [but not 12(R)-HETE] enhance tumor cell adhesion to endothelial cells, subendothelial matrix and fibronectin, but not to type IV collagen. TPA-enhanced adhesion can be reduced by lipoxygenase inhibitors but not by cyclooxygenase inhibitors and in addition, stimulated adhesion can be blocked by pretreatment of tumor cells with specific polyclonal or monoclonal antibodies which react against IRGpIIb/IIIa. 12(S)-HETE-enhanced adhesion can also be inhibited by these same antibodies. In contrast, a lipoxygenase product of linoleic acid, 13(S)-hydroxyoctadecadienoic acid, inhibited TPA and 12(S)-HETE-enhanced tumor cell adhesion to endothelial cells, subendothelial matrix, and fibronectin. These results suggest that (a) IRGpIIb/IIIa is a multifunctional receptor which mediates tumor cell adhesion to a variety of biological substrata, (b) TPA enhances surface expression and/or activation of this receptor possibly via a lipoxygenase metabolite of arachidonic acid, and (c) these effects are opposed by a lipoxygenase metabolite of linoleic acid.     

The lipoxygenase metabolite 12(S)-HETE promotes alpha IIb beta 3 integrin-mediated tumor-cell spreading on fibronectin.

Tumor-cell interaction with the vessel wall during metastasis involves adhesion, induction of endothelial-cell retraction and spreading on the exposed sub-endothelial matrix. The signals for initiation of tumor-cell spreading and the receptors involved are unknown. A protocol was developed to distinguish between initial tumor-cell (B16 amelanotic melanoma; B16a) adhesion to and spreading on fibronectin. The time for maximum spreading was 50 min. Treatment with a lipoxygenase metabolite of arachidonic acid [12(S)-HETE] resulted in maximum spreading in 15 min (max. effect approx. 0.1 microM). Other lipoxygenase metabolites were ineffective. 12(S)-HETE treatment induced a rearrangement of F-actin, vinculin, vimentin intermediate filaments and integrin alpha IIb beta 3, but not integrin alpha 5 beta 1. Antibodies to alpha IIb beta 3 but not alpha 5 beta 1 blocked the 12(S)-HETE effect on B16a spreading. B16a-cell attachment to fibronectin resulted in increased metabolism of arachidonic acid to 12(S)-HETE, which was inhibited by lipoxygenase but not by cyclo-oxygenase inhibitors. Accordingly, lipoxygenase inhibitors but not cyclo-oxygenase inhibitors blocked spontaneous B16a-cell spreading. The protein-kinase-C inhibitors calphostin C, H7 and staurosporine also inhibited spreading, while the protein-kinase-A inhibitor H8 was ineffective. These data suggest that B16a-cell spreading on fibronectin is initiated by a lipoxygenase metabolite [12(S)-HETE] of arachidonic acid and is mediated by protein kinase C.     

Mechanism of the anti-tumour effect of 2,3,5-trimethyl-6-(3-pyridylmethyl) 1,4-benzoquinone (CV-6504).

2,3,5-Trimethyl-6-(3-pyridylmethyl) 1,4-benzoquinone (CV-6504), an inhibitor of 5-lipoxygenase, effectively suppressed growth of the MAC16 tumour in vivo and prevented the accompanying cachexia, when administered daily at a dose of 10 mg kg(-1). There was a reduction in the tumour concentration of linoleic (LA), arachidonic (AA), oleic, stearic and palmitic acid. In order to elucidate the mechanism of the anti-tumour action, the effect of CV-6504 on the metabolism of AA through the 5-, 12- and 15-lipoxygenase pathways has been determined in cell lines sensitive (MAC16, MAC13, MAC26 and Caco-2) and resistant (A549 and DU-145) to CV-6504. Incubation of all cell lines with [3H]AA led to the appearance of [3H]5-, 12- and 15-HETE. Preincubation of MAC16, MAC13, MAC26 and Caco-2 with 10 microM CV-6504 inhibited the conversion of AA to 5-, 12- and 15-HETE, while in A549 and DU-145 cells there was no effect on metabolism through any lipoxygenase pathway. Two other cell lines, MDA-MB-231 and PC-3, sensitive to growth inhibition by CV-6504, are known to require LA for growth, while DU-145, which was insensitive to growth inhibition by CV-6504, showed no growth response to LA. These results suggest that some tumours are dependent on lipoxygenase metabolites of LA and AA for their continual growth, and interference with this pathway produces a specific growth inhibition.     

Metabolism and pharmacokinetics of the anti-tumour agent 2,3,5-trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504).

2,3,5-Trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504) is an effective inhibitor of the growth of established murine adenocarcinomas (MACs) and is shortly to enter clinical investigation. When administered to mice bearing the MAC16 tumour, CV-6504 rapidly disappeared from the plasma and tissues and there was an accumulation of the sulphate and glucuronide metabolites. After 24 h, the concentration of free CV-6504 in the tumour (3.3 microM) was higher than that in the liver (0.24 microM) and equal to the IC50 value for the inhibition of the growth of MAC16 cells in vitro (3 microM). The concentration of glucuronide and sulphate metabolites in both tumour and liver decreased with time. Both the MAC16 tumour and the liver possessed similar beta-glucuronidase activity, which could account for the accumulation of free CV-6504. Although the sulphate and glucuronide conjugates of CV-6504 were ineffective inhibitors of the growth of MAC13 cells in vitro at concentrations up to 100 microM, in vivo at a concentration of 50 mg kg-1 day-1 the conjugates produced a similar anti-tumour effect to CV-6504 at a concentration of 5 mg kg-1 day-1. The MAC13 tumour possessed both beta-glucuronidase and sulphatase activity capable of converting the sulphate and glucuronide conjugates to free CV-6504. Using MAC13 cells ex vivo, CV-6504 inhibited conversion of arachidonic acid to 5-, 12- and 15-hydroxyeicosatetraenoic acids (HETE). The percentage reduction in formation of 12- and 15-HETE exceeded that of 5-HETE. Inhibition of HETE formation may be responsible for the anti-tumour activity of CV-6504.     

血小板型12-脂加氧酶与肿瘤

花生四烯酸通过脂加氧酶(lipoxygenase,LOX)途径代谢产生多种生物活性脂类,这些脂类在炎症、血栓形成和肿瘤演进过程中发挥着重要的作用。LOX按照组织分布被分为5- 、8- 、12 -、15- LOX4 个家族,血小板型12 LOX是LOX的一种,催化花生四烯酸转化为廿碳四烯酸。血小板型12 -LOX在正常组织中主要分布在血小板、人皮肤表皮细胞细胞质和微粒体中。近来研究发现,在多种肿瘤组织和肿瘤细胞系中不仅可检测到血小板型12- LOX的表达,且随着肿瘤级别的增高,瘤细胞表达增强。血小板型12 -LOX催化的代谢产物,如12(S) 羟廿碳四烯酸,已被证明涉及肿瘤细胞凋亡、肿瘤血管形成,因此血小板型12 LOX也成为肿瘤治疗的潜在目标。多项研究表明,血小板型12 -LOX的抑制剂能有效抑制肿瘤,如去甲二氢愈创木酸能有效抑制胶质瘤。对血小板型12- LOX与肿瘤细胞增殖、凋亡和血管生成之间的关系以及其机制进行讨论,另外,对血小板型12- LOX抑制剂预防和治疗肿瘤予以综述。     

15(S)-hydroxyeicosatetraenoic acid induces angiogenesis via activation of PI3K-Akt-mTOR-S6K1 signaling

To determine whether the lipoxygenase metabolites of arachidonic acid, 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acids [5(S)-HETE, 12(S)-HETE, and 15(S)-HETE, respectively] are angiogenic, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All three HETEs stimulated HDMVEC tube formation and migration. Because 15(S)-HETE was found to be more potent than 5(S)-HETE and 12(S)-HETE in HDMVEC tube formation, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 15(S)-HETE stimulated Akt and S6K1 phosphorylation in HDMVEC in a time-dependent manner. Wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3K), blocked both Akt and S6K1 phosphorylation, whereas rapamycin, a specific inhibitor of Akt downstream effector, mammalian target of rapamycin (mTOR), suppressed only S6K1 phosphorylation induced by 15(S)-HETE suggesting that this eicosanoid activates the PI3K-Akt-mTOR-S6K1 signaling in HDMVEC. Wortmannin, LY294002, and rapamycin also inhibited 15(S)-HETE-induced HDMVEC tube formation and migration. In addition, all three HETEs stimulated angiogenesis as measured by in vivo Matrigel plug assay with 15(S)-HETE being more potent. Pharmacologic inhibition of PI3K-Akt-mTOR-S6K1 signaling completely suppressed 15(S)-HETE-induced in vivo angiogenesis. Consistent with these observations, adenoviral-mediated expression of dominant-negative Akt also blocked 15(S)-HETE-induced HDMVEC tube formation and migration and in vivo angiogenesis. Together, these results show for the first time that 15(S)-HETE stimulates angiogenesis via activation of PI3K-Akt-mTOR-S6K1 signaling.     

Studies on the role of platelet eicosanoid metabolism and integrin αIIbβ3 in tumor-cell-induced platelet aggregation

Platelet eicosanoid metabolism resulting from tumor-cell-induced platelet aggregation (TCIPA) was examined in a homologous in vitro system. Rat Walker 256 carcinosarcoma cells induced the aggregation of rat platelets via a thrombin-dependent mechanism with concomitant production of eicosanoid metabolites (e.g., 12-HETE, TXA₂. TCIPA was dependent on the concentration of tumor cells inducing aggregation, as well as cyclooxygenase and lipoxygenase products. Cyclooxygen-ase inhibitors, but not lipoxygenase inhibitors, blocked platelet aggregation induced in vitro by a low concentration of agonist. At a high agonist concentration, neither cyclooxygenase nor lipoxygenase inhibitors alone affected platelet aggregation; however, the combined inhibition of both the cyclooxygenase and lipoxygenase pathways resulted in subsequent inhibition of platelet aggregation regardless of agonist concentration. The extent of platelet TXA₂ and 12-HETE biosynthesis was likewise dependent on and correlated with agonist concentration. The inhibitors used in this study did not significantly inhibit protein kinase C activity at the doses tested. Platelet surface glycoproteins α_IIbβ₃ play an important role in platelet aggregation. The effect of platelet cyclooxygenase and lipoxygenase inhibition in regulating α_IIbβ₃ surface expression was examined by flow cytometric analysis. Thrombin stimulation of washed rat platelets resulted in significantly increased surface expression of platelet α_IIbβ₃ integrin complex. The enhanced surface expression was not inhibited by a cyclooxygenase inhibitor (aspirin), a thromboxane synthase inhibitor (CGS-14854) or a throm-boxane receptor antagonist (SQ 29,548), nor was it stimulated by a thromboxane A₂ mimic (pinane-thromboxane A₂). However, α_IIbβ₃ expression was blocked by lipoxygenase inhibition and stereospecifically increased by the platelet lipoxygenase metabolite 12(S)-HETE. These results suggest that both the platelet lipoxygenase and cyclooxygenase pathways are important for TCIPA but that different mechanisms of action are involved.     

Specific inhibition of phorbol diester-induced granulocyte-macrophage progenitor cell (GM-CFU) differentiation by lipoxygenase inhibitors

The induction of differentiation of SENCAR murine granulocyte-macrophage precursor cells (GM-CFU) by the tumor-promoting phorbol diester 12-O--tetradecanoyl-phorbol-13-acetate (TPA) was inhibited by agents reported to inhibit specific aspects of arachidonic acid metabolism. These agents included phospholipase A2 inhibitors, and eicosatetraynoic acid (ETYA), a competitive inhibitor of arachidonic acid oxygenases. Whereas inhibitors reported specific for lipoxygenases were also active, no comparable effect was observed for inhibitors of the corresponding cyclooxygenase. These findings are therefore consistent with the hypothesis that induced differentiation of GM-CFU cells is regulated by products of arachidonic acid metabolism formed principally via lipoxygenase activity.     

12/15 lipoxygenase regulation of colorectal tumorigenesis is determined by the relative tumor levels of its metabolite 12-HETE and 13-HODE in animal models

Colorectal cancer (CRC) continues to be a major cause of morbidity and mortality. The arachidonic acid (AA) pathway and linoleic acid (LA) pathway have been implicated as important contributors to CRC development and growth. Human 15-lipoxygenase 1 (15-LOX-1) converts LA to anti-tumor 13-S-hydroxyoctadecadienoic acid (13-HODE)and 15-LOX-2 converts AA to 15-hydroxyeicosatetraenoic acid (15-HETE). In addition, human 12-LOX metabolizes AA to pro-tumor 12-HETE. In rodents, the function of 12-LOX and 15-LOX-1 and 15-LOX-2 is carried out by a single enzyme, 12/15-LOX. As a result, conflicting conclusions concerning the role of 12-LOX and 15-LOX have been obtained in animal studies. In the present studies, we determined that PD146176, a selective 15-LOX-1 inhibitor, markedly suppressed 13-HODE generation in human colon cancer HCA-7 cells and HCA-7 tumors, in association with increased tumor growth. In contrast, PD146176 treatment led to decreases in 12-HETE generation in mouse colon cancer MC38 cells and MC38 tumors, in association with tumor inhibition. Surprisingly, deletion of host 12/15-LOX alone led to increased MC38 tumor growth, in association with decreased tumor 13-HODE levels, possibly due to inhibition of 12/15-LOX activity in stroma. Therefore, the effect of 12/15-LOX on colorectal tumorigenesis in mouse models could be affected by tumor cell type (human or mouse), relative 12/15 LOX activity in tumor cells and stroma as well as the relative tumor 13-HODE and 12-HETE levels.     

Effect of a cancer cachectic factor on protein synthesis/degradation in murine C2C12 myoblasts: modulation by eicosapentaenoic acid.

The effect of a proteolysis inducing factor (PIF) on protein synthesis and degradation and the modulation of this effect by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA), have been examined using a surrogate model system, C2C12 myoblasts in vitro. After 90 min of incubation, PIF produced a significant inhibition of protein synthesis in a dose-dependent manner, with maximal inhibition at a concentration of 4 nM. The effect was attenuated both by treatment with a monoclonal antibody to PIF and by treatment with insulin at physiological concentrations (1 nM) and below (0.1 nM), but not by EPA (50 microM). The inhibitory effect on protein synthesis was transitory and was not seen after prolonged incubation with PIF. An increased rate of protein degradation was observed in C2C12 myoblasts after addition of PIF, which was also maximal at a concentration of PIF of 4 nM. Higher concentrations of PIF did not produce an increase in protein degradation. Unlike the effect on protein synthesis, the enhanced protein degradation was completely abolished by pretreatment with 50 microM EPA, suggesting that the two effects are mediated by different mechanisms. PIF produced an increased release of [3H]arachidonic acid from prelabeled myoblasts with a dose-response curve parallel to that of protein degradation and with a maximum at 4 nM PIF. Release of [3H] arachidonic acid was completely blocked in cells pretreated with 50 microM EPA, suggesting that the effect was related to protein degradation. The [3H]arachidonic acid was rapidly metabolized to prostaglandins E2 and F2alpha and to 5-, 12-, and 15-hydroxyeicosatetraenoic acids (HETEs). Production of all eicosanoids was attenuated in cells pretreated with EPA. Of all of the metabolites, only 15-HETE produced a significant increase in protein degradation in C2C12 myoblasts with a maximal effect at 30 nM and with a bell-shaped dose-response curve similar to that produced by PIF. These results suggest that PIF enhances protein degradation as a result of an increased production of 15-HETE.     

12(S)-hydroxyeicosatetraenoic acid increases the actin microfilament content in B16a melanoma cells: A protein kinase-dependent process

12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], a lipoxygenase metabolite of arachidonic acid, has been shown to be involved in a wide variety of cellular activities (i.e., adhesion, spreading, motility, invasion) which promote metastasis to occur in tumor cells. In this study, several techniques (Western blotting, flow cytometry and DNase I assay) were performed to examine the alterations in the distribution of G- and F-actin expressed in B16a melanoma cells. Each of these methods independently revealed that 12(S)-HETE treatment (0.1 mM, 15 min) resulted in an increase in the F-actin content in the cytoskeletal preparations. Since the integrity of cytoskeletal networks (i.e., actin filaments) can be dynamically regulated through protein phosphorylation, we investigated the potential role of several protein kinases in the 12(S)-HETE-induced actin polymerization. By flow cytometric analysis, 12(S)-HETE was found to increase the actin filament contents. This effect could be inhibited by protein kinase C (PKC) inhibitors (calphostin C and staurosporine) as well as by protein tyrosine kinase (PTK) inhibitor (genistein) but not by protein kinase A inhibitor (H8), suggesting that the 12(S)-HETE effect involves PKC and PTK. This conclusion is consistent with the observations that phorbol 12-myristate-13-acetate (PMA) mimics the biological effect of 12(S)-HETE in promoting the F-actin formation in B16a cells. As a final analysis, direct protein phosphorylation studies indicate that 12(S)-HETE treatment led to enhanced phosphorylation of myosin light chain, which may contribute to the increased stress fiber formation following 12(S)-HETE stimulation. Int. J. Cancer 77:271–278, 1998.© 1998 Wiley-Liss, Inc.     

What are cyclooxygenases and lipoxygenases doing in the driver's seat of carcinogenesis?

Substantial evidence supports a functional role for cyclooxygenase- and lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Genetic intervention studies firmly established cause-effect relations for cyclooxygenase-2, but cyclooxygenase-1 may also be involved. In addition, pharmacologic cyclooxygenase inhibition was found to suppress carcinogenesis in both experimental mouse models and several cancers in humans. Arachidonic acid-derived eicosanoid or linoleic acid-derived hydro[peroxy]fatty acid signaling are likely to be involved impacting fundamental biologic phenomena as diverse as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, long chain unsaturated fatty acid oxidation reactions indicate antipodal functions of distinct lipoxygenase isoforms in carcinogenesis, i.e., the 5- and platelet-type 12-lipoxygenase exhibit procarcinogenic activities, while 15-lipoxygenase-1 and 15-lipoxygenase-2 may suppress carcinogenesis.     

Alterations in lipoxygenase and cyclooxygenase-2 catalytic activity and mRNA expression in prostate carcinoma

Recent studies in prostate tissues and especially cell lines have suggested roles for arachidonic acid (AA) metabolizing enzymes in prostate adenocarcinoma (Pca) development or progression. The goal of this study was to more fully characterize lipoxygenase (LOX) and cyclooxygenase-2 (COX-2) gene expression and AA metabolism in benign and malignant prostate using snap-frozen tissues obtained intraoperatively and mRNA analyses and enzyme assays. Formation of 15-hydroxyeicosatetraenoic acid (15-HETE) was detected in 23/29 benign samples and 15-LOX-2 mRNA was detected in 21/25 benign samples. In pairs of pure benign and Pca from the same patients, 15-HETE production and 15-LOX-2 mRNA were reduced in Pca versus benign in 9/14 (P=.04) and 14/17 (P=.002), respectively. Under the same conditions, neither 5-HETE nor 12-HETE formation was detectable in 29 benign and 24 tumor samples; with a more sensitive assay, traces were detected in some samples, but there was no clear association with tumor tissue. COX-2 mRNA was detected by nuclease protection assay in 7/16 benign samples and 5/16 tumors. In benign and tumor pairs from 10 patients, COX-2 was higher in tumor versus benign in only 2, with similar results by in situ hybridization. Paraffin immunoperoxidase for COX-2 was performed in whole mount sections from 87 additional radical prostatectomy specimens, with strong expression in ejaculatory duct as a positive control and corroboration with in situ hybridization. No immunostaining was detected in benign prostate or tumor in 45% of cases. Greater immunostaining in tumor versus benign was present in only 17% of cases, and correlated with high tumor grade (Gleason score 8 and 9 vs. 5 to 7). In conclusion, reduced 15-LOX-2 expression and 15-HETE formation is the most characteristic alteration of AA metabolism in Pca. Increased 12-HETE and 5-HETE formation in Pca were not discernible. Increased COX-2 expression is not a typical abnormality in Pca in general, but occurs in high-grade tumors.