Constitutive expression of 8-lipoxygenase in papillomas and clastogenic effects of lipoxygenase-derived arachidonic acid metabolites in keratinocytes

The expression pattern, enzymatic activity, and products of 8-lipoxygenase (LOX) were analyzed in normal and neoplastic skin of NMRI mice. While barely detectable in normal epidermis, 8-LOX was transiently induced by 12-O-tetradecanoylphorbol-13-acetate and constitutively expressed in papillomas but not carcinomas obtained by the initiation-promotion protocol of mouse skin carcinogenesis. The product profile and chirality of both the native and the recombinant protein produced the S enantiomers of 8-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid (8-HETE) and 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE) as the main arachidonic acid- and linoleic acid-derived metabolites. As compared with normal epidermis, papillomas exhibited 25- and 4-fold elevated levels of 8-HETE and 9-HODE, respectively. However, the varying S to R ratios of 8-HETE and the predominance of 9(R)-HODE indicated that in addition to 8(S)-LOX, other enzymes yet to be defined may be involved in 8-HETE and 9-HODE production. The massive accumulation of both 8-HETE and 12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HETE) point to a critical role of these LOX pathways in epidermal tumor development, in particular in the papilloma stage. Here we showed that 8- and 12-hydroperoxyeicosatetraenoic acids and 8- and 12-HETE induce chromosomal alterations in cycling primary basal keratinocytes.     

Metabolic suppression of platelet-type 12-lipoxygenase in human uterine cervix with invasive carcinoma

Several types of lipoxygenases with various functions occur in mammalian cells. Although the presence of 12-lipoxygenase activity has been reported in uterine tissues, neither its type nor its biological functions have yet been established. Moreover, the putative role of uterine 12-lipoxygenase in cervical cancer has not been addressed before. Homogenates of uterine tissues from women without cancer and from patients with invasive cervical carcinoma were incubated with (1-(14)C)-arachidonic acid under various conditions and the labelled reaction products were analyzed both by thin-layer chromatography and by high-pressure liquid chromatography. 12-Lipoxygenase protein was estimated by Western blot using anti-serum against recombinant human platelet-type 12-lipoxygenase. Highest concentrations and activities of 12-lipoxygenase were found in the exocervix. The formation of 12S-hydroxy-5Z,8Z,10E, 14Z-eicosatetraenoic acid (12-HETE) was stimulated by micromolar concentrations of 13S-hydroperoxy-9Z,11E-octadecadienoic acid, suggesting metabolic control of the 12-lipoxygenase activity via the hydroperoxide tone. Immunohistochemical investigation revealed that the enzyme is mainly located in the squamous epithelium, and is of platelet-type. Significantly lower values for the 12-HETE formation were found in samples from patients with invasive cervical carcinoma, whereas the amount of immunochemically detectable 12-lipoxygenase protein was unaltered. At the same time the expression levels of the bcl-2 gene were enhanced. Thus, it is concluded that during carcinogenesis the hydroperoxide-reducing capacity of the uterine cervix tissue is enhanced, possibly mediated by bcl-2 protein, and in turn metabolically suppresses the 12-lipoxygenase activity. Furthermore, the data suggest an anti-carcinogenic action of 12-lipoxygenase in human cervix, in contrast to its reported pro-carcinogenic action in breast cancer.     

Overexpression of leukocyte-type 12-lipoxygenase promotes W256 tumor cell survival by enhancing alphavbeta5 expression

The metabolism of arachidonic acid (AA) leads to the generation of biologically active metabolites that have been implicated in cell growth and proliferation, as well as survival and apoptosis. We have previously demonstrated that rat Walker 256 (W256) carcinosarcoma cells express the platelet-type 12-lipoxygenase (12-LOX) and synthesize 12(S)- and 15(S)-HETE as their major LOX metabolites. Here we show that Walker 256 cells also express leukocyte-type 12-LOX and that its overexpression in these cells significantly extends their survival and delays apoptosis when cells are cultured under serum-free conditions. Under serum-free conditions, the expression of leukocyte-type 12-LOX is upregulated. 12-LOX-transfected W256 cells had a more spread morphology in culture compared with wild-type or mock-transfected cells. Examination of W256 cells showed that the cells expressed a number of integrins on their surface. Overexpression of 12-LOX enhanced the surface expression and focal adhesion localization of integrin alphavbeta5, while not affecting other integrins. Also, the 12-LOX-transfected W256 cells exhibited higher levels of microfilament content. Treatment of cells with monoclonal antibody to alphavbeta5 or cytochalasin B (a microfilament-disrupting agent), but not antibodies to other integrin receptors, resulted in significant apoptosis, characterized by rapid rounding up and detachment from the substratum. These results show that the 12-LOX pathway is a regulator of cell survival and apoptosis, by affecting the expression and localization of the alphavbeta5 integrin and actin microfilaments in Walker 256 cells.     

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。     

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.     

Diverse roles of 2-arachidonoylglycerol in invasion of prostate carcinoma cells: Location, hydrolysis and 12-lipoxygenase metabolism

Endogenous 2-arachidonoylglycerol (2-AG) is antiinvasive in androgen-independent prostate carcinoma (PC-3) cells. Invasion of PC-3 cells is also inhibited by exogenously added noladin ether, a non-hydrolyzable analog of 2-AG. In contrast, exogenous 2-AG has the opposite effect. Cell invasion significantly increased with high concentrations of exogenous 2-AG. In PC-3 cells, arachidonic acid (AA) and 12-hydroxyeicosatetraenoic acid (12-HETE) concentrations increased along with exogenously added 2-AG, and 12-HETE concentrations increased with exogenously added AA. Invasion of PC-3 cells also increased with exogenously added AA and 12(S)-HETE but not 12(R)-HETE. The exogenous 2-AG-induced invasion of PC-3 cells was inhibited by 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP, an inhibitor of 2-AG hydrolysis) and baicalein (a 12-LO inhibitor). Western blot and RT-PCR analyses indicated expression of 12-HETE producing lipoxygenases (LOs), platelet-type 12-LO (P-12-LO) and leukocyte-type 12-LO (L-12-LO), in PC-3 cells. These results suggest that exogenous 2-AG induced, rather inhibited, cell invasion because of its rapid hydrolysis to free AA, and further metabolism by 12-LO of AA to 12(S)-HETE, a promoter of PC cell invasion. The results also suggest that PC-3 cells and human prostate stromal (WPMY-1) cells released free AA, 2-AG, and 12-HETE. In the microenvironment of the PC cells, this may contribute to the cell invasion. The 2-AG hydrolysis and concentration of 2-AG in microenvironment are critical for PC cell's fate. Therefore, inhibitors of 2-AG hydrolysis could potentially serve as therapeutic agents for the treatment of prostate cancer. (c) 2007 Wiley-Liss, Inc.     

Generation of the arachidonic acid metabolite 8-HETE by extracts of mouse skin treated with phorbol ester in vivo; identification by 1H-n.m.r. and GC-MS spectroscopy

After a single in vivo application of 20 nmol of the ‘complete’tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) orof the ‘incomplete’ promoter 12-O-retinoylphorbol-13-acetate(RPA) to the back skin of mice, a major arachidonic acid metabolite(AAMX) was found in cell-free epidermal preparations after additionof arachidonic acid (AA). Studies with cyclooxygenase and lipoxygenaseinhibitors indicated that this metabolite is a lipoxygenaseproduct. The metabolite has been purified by sequential useof t.l.c. and h.p.l.c. and identified as 8-hydroxy-5Z, 9E, 11Z,14Z-eicosatetraenoic acid (8-HETE) by means of GC-MS and ¹H-n.m.r.spectroscopy. To assist in the characterization of AAMX, a referencemixture of 8-HETE and 9-HETE was used for which a complete structuralanalysis could be performed using one- and twodimensional ¹H-n.m.r.at 500 MHz. The production of 8-HETE has been shown to startwith a lag phase of 3 h after TPA treatment and to reach a maximumafter 24–48 h. Nonpromoting phorbol esters are 10-fold,the Ca²⁺-ionophore A 23187 100-fold, less efficient in inducingin vivo the lipoxygenase responsible for 8-HETE production.3-Day-old neonatal mice cannot be induced to generate 8-HETEin response to TPA, whereas 8-day-old mice show an extremelystrong response. Neither primary basal keratinocytes, isolatedfrom TPA-treated mouse epidermis nor TPA-treated epidermal celllines generate 8-HETE, indicating that the metabolite may notoriginate from these epithelial cells.     

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.     

Prostaglandin and hydroxyeicosatetraenoic acid synthesis by human mesenchymal tumors

The metabolism of arachidonic acid was investigated by radioimmunoassay and chromatographic techniques in 5 sarcomas and one embryonal carcinoma of human origin maintained as transplantable tumors in nude mice. The results obtained indicate that: the absolute quantities of arachidonic acid metabolites produced by a given tumor varied between experiments but the overall distribution pattern of these products, in general, remained constant from passage to passage; each tumor showed a different arachidonic acid metabolite profile in quality and quantity; 2 sarcomas of the same histological type could be clearly distinguished by their arachidonic acid metabolites; the predominant product in all tumors was 12-HETE or 15-HETE, whereas thromboxane A2 was synthesized in low quantities by all tumors; PGF2 alpha was synthesized at the highest rate by an alveolar rhabdomyosarcoma; PGE2 synthesis was highest in a malignant fibrous histiocytoma; and total prostaglandin synthesis was low in the chondrosarcoma and synovial-cell sarcomas. All results reported in this study are for the complete tumor which includes both neoplastic and stromal cells. The role that these products play in the biological behavior of mesenchymal tumor cells and normal tissues of the host remains to be determined.     

Effect of 12-O-tetradecanoylphorbol-13-acetate on inhibition of expression of keratin 1 mRNA in mouse keratinocytes mimicked by 12(S)-hydroxyeicosatetraenoic acid

Differentiation of cultured keratinocytes is controlled by the calcium concentration of the medium and is marked by the expression of differentiation-specific keratins. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) alters the normal differentiation program and suppresses keratin (K) 1 expression. Based on reported similarities in the effects of TPA and the arachidonic acid metabolite 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), we hypothesized that 12(S)-HETE might suppress K1 expression in mouse keratinocytes. We also investigated the effect of pretreatment with 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE) because others have reported that 13(S)-HODE prevents 12(S)-HETE–induced events. In our study, 100 nM 12(S)-HETE mimicked the effect of 500 nM TPA in suppressing K1 mRNA expression within 24 h of calcium-induced differentiation. Pretreatment with 100 nM 13(S)-HODE blocked the 12(S)-HETE effect but not the TPA effect. A role for protein kinase C (PKC) was suggested for both TPA and 12(S)-HETE based on the loss of response with the PKC inhibitors bryostatin-1 or RO-31-8220. Both TPA and 12(S)-HETE stimulated keratinocyte PKC activity. Pretreatment with 13(S)-HODE blocked the 12(S)-HETE–induced increase in PKC activity. Immunoblotting showed that whereas TPA caused a rapid, partial translocation of the PKCα isozyme, it had no effect on the distribution of PKCδ. Conversely, 12(S)-HETE had no effect on the distribution of PKCα but caused a complete translocation of PKCδ. Pretreatment with 13(S)-HODE prevented 12(S)-HETE–elicited translocation of PKCδ. We conclude that 12(S)-HETE mimics the effect of TPA on K1 mRNA and that the effect is mediated through different isoforms of PKC. Mol. Carcinog. 19:157–164, 1997. © 1997 Wiley-Liss, Inc.     

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.     

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.     

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.     

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.     

Inhibition of human malignant neuroblastoma cell DNA synthesis by lipoxygenase metabolites of arachidonic acid

In vivo studies have shown that inhibitors of cyclooxygenase metabolism of arachidonic acid may diminish growth and metastasis of certain tumors. Because cyclooxygenase inhibition may increase the production of lipoxygenase products of arachidonic acid metabolism, we have investigated the effect of two such products, 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) on tumor cell proliferation in vitro. When neuroblastoma cells (SK-N-SH) in culture were treated with 12-HETE for 18 hr, incorporation of [3H]thymidine was inhibited up to 64% at concentrations from 20 to 50 microM. Under the same conditions, 15-HETE resulted in inhibition of up to 46%, while arachidonic acid had no apparent effect. When evaluated in the presence of serum, 12-HETE at a concentration of 120 microM produced a 20.6 +/- 2.8% (S.E.) inhibition of the increase in total DNA content over 48 hr, while 15-HETE at this concentration produced a 16.5 +/- 5.3% inhibition. We conclude that 12-HETE, the product of platelet lipoxygenase, and 15-HETE, a product of neutrophil and lymphocyte lipoxygenases, can inhibit human neuroblastoma cell growth in vitro and may play a role in the effect of cyclooxygenase inhibitors on tumor growth in vivo.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-mediated epidermal ornithine decarboxylase induction and skin tumor promotion by new lipoxygenase inhibitors lacking protein kinase C inhibitory effects

Both 2,3,5-trimethyl-6-(12-hydroxy-5, 10-dodecadiynyl)-l, 4-benzoquinone(AA861) and 3, 4, 2‘, 4’-tetrahydroxychalcone inhibited12-lipoxygenase of mouse epidermis. The IC₅₀ of AA861 and 3,4, 2‘, 4’-tetrahydroxychalcone for epidermal 12-lipoxygenasewere 1.9 and 0.2 µM, respectively. These agents showedvery weak inhibitory actions on epidermal cyclooxygenase, withthe potency of inhibition for cydooxy-genase less than 1/50of that for lipoxygenase. Induction of epidermal ornithine decarboxylaseby 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 10 nmol/mouse)was potently inhibited by these agents in a dose-dependent manner(1–30µmol/mouse). TPA (5 nmol/mouse)-induced skintumor formation was also strongly suppressed by these agents(15 µmol/mouse). Both AA861 and 3, 4, 2‘, 4’-tetrahydroxy-chalconefailed to inhbit partially purified epidermal protein kinaseC activity. These results support the proposed involvement oflipoxygenase product(s) of arachidonic acid in TPA-induced skintumor promotion.     

Expression, subcellular localization and putative function of platelet-type 12-lipoxygenase in human prostate cancer cell lines of different metastatic potential

The involvement of 12-lipoxygenase (12-LOX) expression and function in tumor metastasis has been demonstrated in several murine tumor cell lines. In addition, 12-LOX expression was detected in human prostatic tumors and correlated to the clinical stage of disease. Here we provide data that human prostate cancer cell lines express the platelet-type isoform of 12-LOX at both the mRNA and protein levels, and immunohistochemistry revealed 12-LOX expression in human prostate tumors. The enzyme was localized to the plasma membrane, cytoplasmic organelles and nucleus in non-metastatic cells (PC-3 nm) and to the cytoskeleton and nucleus in metastatic cells (DU-145). After orthotopic/intraprostatic injection of tumor cells into SCID mice, the metastatic prostate carcinoma cells (DU-145) expressed 12-LOX at a significantly higher level compared with the non-metastatic counterparts, PC-3nm. The functional involvement of 12-LOX in the metastatic process was demonstrated when DU-145 cells were pretreated in vitro with the 12-LOX inhibitors N-benzyl-N-hydroxy-5-phenylpentamide (BHPP) or baicalein, the use of which significantly inhibited lung colonization. These data suggest a potential involvement of 12-LOX in the progression of human prostate cancer.