lipoxygenase inhibitors as potential cancer chemopreventives · a cysteine protease involved in...

Review

Lipoxygenase Inhibitors as Potential Cancer Chemopreventives

Vernon E. Steele,1 Cathy A. Holmes, Ernest T. Hawk,Levy Kopelovich, Ronald A. Lubet, James A. Crowell,Caroline C. Sigman, and Gary J. KelloffChemoprevention Branch, Division of Cancer Prevention, National CancerInstitute, Bethesda, Maryland 20892 [V. E. S., E. T. H., L. K., R. A. L.,J. A. C., G. J. K.], and CCS Associates, Mountain View, California 94043[C. A. H., C. C. S.]

AbstractMounting evidence suggests that lipoxygenase (LO)-catalyzed products have a profound influence on thedevelopment and progression of human cancers.Compared with normal tissues, significantly elevatedlevels of LO metabolites have been found in lung,prostate, breast, colon, and skin cancer cells, as well as incells from patients with both acute and chronicleukemias. LO-mediated products elicit diverse biologicalactivities needed for neoplastic cell growth, influencinggrowth factor and transcription factor activation,oncogene induction, stimulation of tumor cell adhesion,and regulation of apoptotic cell death. Agents that blockLO-catalyzed activity may be effective in preventingcancer by interfering with signaling events needed fortumor growth. In fact, in a few studies, LO inhibitorshave prevented carcinogen-induced lung adenomas andrat mammary gland cancers.

During the past 10 years, pharmacological agentsthat specifically inhibit the LO-mediated signalingpathways are now commercially available to treatinflammatory diseases such as asthma, arthritis, andpsoriasis. These well-characterized agents, representingtwo general drug effect mechanisms, are considered goodcandidates for clinical chemoprevention studies. Onemechanism is inhibition of LO activity (5-LO andassociated enzymes, or 12-LO); the second is leukotrienereceptor antagonism. Although the receptor antagonistshave high potential in treating asthma and other diseaseswhere drug effects are clearly mediated by theleukotriene receptors, enzyme activity inhibitors may bebetter candidates for chemopreventive intervention,because inhibition of these enzymes directly reduces fattyacid metabolite production, with concomitant damping ofthe associated inflammatory, proliferative, and metastaticactivities that contribute to carcinogenesis. However,because receptor antagonists have aerosol formulationsand possible antiproliferative activity, they may also have

potential, particularly in the lung, where topicalapplication of such formulations is feasible.

IntroductionEicosanoids derived from the arachidonic acid cascade havebeen implicated in the pathogenesis of a variety of humandiseases, including cancer, and are now believed to play im-portant roles in tumor promotion, progression, and metastaticdisease. Although most attention has focused on PGs2 and otherCOX-derived metabolites, mounting evidence suggests thatLO-catalyzed products, LTs, and HETEs also exert profoundbiological effects on the development and progression of hu-man cancers. For example, 12-LO mRNA expression has beenwell-documented in many types of solid tumor cells, includingthose of prostate, colon, and epidermoid carcinoma (1, 2). Also,12(S)-HETE3 production by some tumor cells, including pros-tate cells, has been positively correlated to their metastaticpotential (3, 4). Also, studies show that 12(S)-HETE is a criticalintracellular signaling molecule, stimulating PKC and elicitingthe biological actions of many growth factors and cytokines thatregulate transcription factor activation and induction of onco-genes or other gene products needed for neoplastic cell growth(5–7). Some of these include EGF, FGF, PDGF, tumor necrosisfactor, granulocyte-macrophage colony-stimulating factor, andboth IL-1 and IL-3 (8–12). Additionally, PKC activation by12(S)-HETE mediates the release and secretion of cathepsin B,a cysteine protease involved in tumor metastasis and invasion,particularly in colon cancer cells (13). Furthermore, tumor cellsynthesis of 12(S)-HETE stimulates adhesion by increasing thesurface expression of integrin receptors (14, 15). Besides 12(S)-

Received 10/16/98; revised 3/2/99; accepted 3/10/99.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby markedadvertisementinaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.1 To whom requests for reprints should be addressed, at National Cancer Institute,Division of Cancer Prevention, Chemoprevention Branch, EPN 201E, MSC 7322,9000 Rockville Pike, Bethesda, MD 20892-7322.

2 The abbreviations used are: PG, prostaglandin; COX, cyclooxygenase; LO,lipoxygenase; LT, leukotriene; HETE, hydroxyeicosatetraenoic acid; PKC, pro-tein kinase C; EGF, epidermal growth factor; FGF, fibroblast growth factor;PDGF, platelet-derived growth factor; IL, interleukin; PLA2, phospholipase A2;FLAP, 5-LO activating protein; PMN, polymorphonuclear neutrophil; HODE,hydroxyoctadecadienoic acid; GRP, gastrin-releasing peptide; IGF, insulin-likegrowth factor; NDGA, nordihydroguaiaretic acid; BHPP,N-benzyl-N-hydroxy-5-phenylpentanamide; TGF, transforming growth factor; MNU,N-nitrosomethy-lurea; DMBA, 7,12-dimethylbenz(a)anthracene; TPA, phorbol 12-myristate 13-acetate; ODC, ornithine decarboxylase; FDA, Food and Drug Administration;ZD2138, 6-((fluoro-5-(tetrahydro-4-methoxy-2H-pyran-4-yl)phenoxy)methyl)-1-methyl-2(1H)-quinolinone; MK-886, 3-(1(9p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl)-2,2-dimethylpropanoic acid; MK0591, 3-(1((4-chloro-phenyl)methyl)-3-((1,1-dimethylethyl)thio)-5-(quinolin-2-ylmethyloxy)-1H-indol-2yl)-2,2-dimethyl-propanoate; BAY-X1005, (R)-2-(4-(quinolin-2-yl-methoxy)phenyl)-2-cyclopentyl acetic acid; SC 41930, 7-(3-(4-acetyl-3methoxy-2-propylphenoxy)propoxy)-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylicacid; f-MLP, N-formyl-l-methionyl-l-leucyl-l-phenylalanine; SC 53228, 7-(3-(2-(cyclopropylmethyl)-3-methoxy-4-((methylamino)carbonyl)phenoxy)propoxy)-3,4-dihydro-8-propyl-2H-1-benzopyran-2-propanoic acid; esculetin, 6,7-dihydroxy-2H-1-benzopyran-2-one; MAC, murine colon adenocarcinoma;baicalein, 5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one; ACF, aberrantcrypt foci.3 Throughout this manuscript, references are made to studies of 12-HETE. If it isclear from the published study report that the form is 12(S)-HETE, it is sodesignated. If the form is not specified or cannot otherwise be determined, thecompound is cited simply as 12-HETE. Probably many of the 12-HETEs cited areactually 12(S)-HETE. We apologize for this unavoidable lack of clarity.

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HETE, other LO metabolites, particularly the 5-LO products(5-HETEs), have been implicated in cancer development. Forexample, data published recently show that 5-HETE directlystimulates prostate cancer cell growth (16). Like 12(S)-HETE,these molecules are capable of exerting pleiotropic effects onnormal and malignant cells through autocrine- and paracrine-mediated mechanisms. Taken together, these data imply thatLO products, particularly those of 5- and 12-LO, may playimportant roles in modulating cancer development. On the basisof this information, pharmaceutical agents that directly interferewith the production of LO metabolites or antagonize the sig-naling functions of LO products may be effective in preventingcancer. Table 1 lists potential chemopreventive mechanismsassociated with inhibition of the LO pathway.

Over the past 10 years, pharmacological agents that spe-cifically inhibit the LO metabolic pathway have been developedto treat inflammatory diseases such as asthma, ulcerative colitis,arthritis, and psoriasis. As inflammatory mediators, LTs elicit anumber of reactions, including vessel wall adhesion, smoothmuscle contraction, granulocyte degranulation, chemotaxis,and increased mucous secretion and vascular permeability (17).These agents include 5-LO inhibitors of theN-hydroxyureaseries developed by Abbott [Leutrol, Zyflo (Zileuton), andABT-761] and, among others, the redox inhibitor AA-861(Takeda Chemical), the non-redox inhibitor ZD2138 (Zeneca),agents that interact with the FLAP, including MK-0591(Merck) and BAY-X1005 (Bayer), and LTB4 receptor antago-nists SC 41930 (Searle), Accolate (zafirlukast; Zeneca), Singu-lair(montelukast; Merck), and Ultair (pranlukast; Ono/Smith-Kline Beecham). Baicalein and esculetin are among severalexperimental compounds demonstrating 12-LO inhibitory ac-tivity that may show promise as antiproliferative agents. Thisreport discusses the current clinical status of these and otheragents and methods to assess their cancer chemopreventivepotential and offers an overview of the LO biosynthetic path-way, its metabolic products, and their significance in humandisease and the development of cancer.

LO Biochemistry and Molecular BiologyThe LOs comprise a family of non-heme iron-containing di-oxygenases that catalyze the stereospecific oxygenation of the5-, 12-, or 15-carbon atoms of arachidonic acid (17–19). Incells, arachidonic acid is esterified to membrane phospholipidsin the sn2 position. As depicted in Fig. 1, the reaction beginswith the intracellular release of arachidonic acid, mediated by

either PLA2, or by the combined actions of phospholipase Cand diacylglycerol kinase or phospholipase D and PLA2 (18).In leukocytes, cytokines including IL-1 and tumor necrosisfactor can activate PLA2 by stimulating a phospholipase-acti-vating protein (20). Once released, arachidonic acid is eitherconverted by catalytic action of 5-, 12-, or 15-LOs into thecorresponding HETEs or is metabolized into LTs or lipoxinsthrough additional sequential reactions, which depend on thebiosynthetic capacity of each specific cell type (see below).

LT production proceeds via 5-LO, which catalyzes thefirst two steps in the metabolic cascade. 5-LO in the presenceof FLAP catalyzes the oxygenation of arachidonic acid into5-hydroperoxyeicosatetraenoic acid, followed by a second re-action in which 5-hydroperoxyeicosatetraenoic acid is dehy-drated to form the epoxide LTA4. Once formed, LTA4 is furthermetabolized to either LTB4 via stereoselective hydration byLTA4 hydrolase or to LTC4 through glutathione conjugationcatalyzed by LTC4 synthase. Sequential metabolic reactions,catalyzed byg-glutamyl transferase and a specific membrane-bound dipeptidase, convert LTC4 into LTD4 and LTE4, respec-tively. These three sulfidopeptide LTs are commonly referredto as the slow-reacting substances of anaphylaxis (21). In thelung, sulfidopeptide LTs are known to act on a single high-affinity, smooth muscle receptor, thecys-LT1 receptor (22),resulting in bronchoconstriction and alterations in vascular per-meability and mucous secretion in this tissue (23). Importantcellular sources of these LTs include eosinophils, mast cells,and basophils (24).

The intracellular localization of 5-, 12-, and 15-LO pro-teins varies depending on the enzyme and specific cell type(25). In human PMNs, 5-LO was initially reported to be asoluble, cytosolic protein (26, 27). However, later studiesshowed that, upon activation with Ca21, 5-LO translocatesfrom the cytosol to the nuclear membrane (28), where it inter-acts with FLAP, anMr 18,000 protein that resides in the nuclearenvelope (29, 30). Indeed, reversible shifting of 5-LO proteinbetween soluble and membrane-bound forms has been con-firmed by other investigators (28, 31), although with differ-ences in distribution patterns and trafficking in various celltypes (32–35). Subcellular localization studies suggest thattranslocation of 12-LO protein from the cytosol to membranealso occurs upon activation (36, 37), whereas 15-LO appears toexist only in cytosol (38, 39).

Metabolic products of the 5-, 12-, or 15-LO biosyntheticpathways modulate the growth of several normal human cells

Table 1 LO inhibition: Potential chemopreventive mechanisms

Chemopreventive mechanism Specific activity

Antiproliferation 12-LO inhibitionModulate signal transduction (5) 2PKC (5–7, 91, 213), cAMP (214, 215), tyrosine kinase (216, 217)Modulate growth factor activity (8–10, 71, 119, 218, 219) 2EGF, FGF, PDGF, tumor necrosis factor, GRP, K-ras, c-myc, int-2Inhibit oncogene expression (94, 137, 220)

Apoptosis induction (155, 221, 222) 5-LO, 12-LO inhibitionAntioxidant activity (145) 2Free radical productionBlock metabolic activation (72, 73)

Angiogenesis inhibition 12-LO inhibitionInhibit membrane degradation (13) 2Cathepsin BDecrease tumor cell adhesion (7, 9) 2Endothelial cell growthDecrease tumor cell motility (223)Inhibit metastasis (2, 4)

Antiinflammatory 5-LO/FLAP inhibitionPrevent tissue damage (224–226) 2PMN activationModulate immune response (227) 2T-lymphocyte activation

468 Review: Lipoxygenase Inhibitors as Chemopreventives



types, including T lymphocytes (40), skin fibroblasts (41),epidermal keratinocytes (42), and glomerular epithelial cells(43). Both LTB4 and LTC4 increasein vitro growth of arterialsmooth muscle cells (44), airway epithelial cells (45), andmitogen-stimulated lymphocytes (46). Besides these cells, LTsplay important roles in regulating both human and murinehematopoiesis (47–50).

The 5-, 12-, and 15-LO genes have been cloned andsequenced in several mammalian species (25, 51). Severalisoforms of 12-LO have been characterized, each with distinctgenomic sequences, gene structure, immunogenicity, and sub-strate specificity (51–53). These include human platelet-type12-LO, referred to as P-12-LO, which metabolizes arachidonicacid into 12(S)-HETE (54); the leukocyte type, or L-12-LO,which converts arachidonic acid or linoleic acid into 12(S)-HETE and also produces small quantities of 15(S)-HETE (55);and 12-LO, a unique epithelial type first isolated from bovinetracheal epithelial cells, which catalyzes the synthesis of both12(S)- and 15(S)-HETE from arachidonic acid (56, 57). How-ever, these classifications are not precise. P-12-LO has alsobeen identified in human keratinocytes and epidermal carci-noma cells lines, and L-12-LO has been found in peritonealmacrophages, trachea epithelium, whole brain, and othersources, although it is not expressed in mouse leukocytes (25).Yamamotoet al. (58) recently reviewed the molecular struc-ture, catalytic properties, and biological activities of the 12-LOisoenzymes.

Mapped to chromosome 10, the human 5-LO gene appearsthe least related to LO genes such as P-12-LO and 15-LO,which are located on chromosome 17 (59). In cDNA compar-isons, L-12-LO shows greater sequence homology (80–90%) to15-LO than does P-12-LO (60–70%; Ref. 60). Genetic analysisfound that all LOs contain a highly conserved region of fivehistidine residues, possible binding sites for non-heme iron(61). Site-directed mutagenesis experiments with human 5-LOfurther showed that two of the five histidine residues are nec-essary for LO catalytic activity (62, 63). To date, no geneticdefects at the 5- or 12-LO locus have been described. Severalreports identified P-12-LO deficiency in myeloproliferativebleeding disorders (64, 65).

Specific factors that regulate LO gene expression havebeen identified. Glucose, EGF, and angiotensin II stimulate12-LO mRNA expression, production of HETEs and HODEs(35, 66), and both IL-4 and IL-13 are positive regulators ofmonocyte 15-LO gene expression (67, 68).

LO Metabolites in CarcinogenesisEvidence from studies in human cancer cells shows that the LOpathways are involved in carcinogenesis in several major tis-sues. More importantly, a few studies in animal carcinogenesismodels show that inhibition of the LO pathways also mayinhibit carcinogenesis.Lung. Two recent studies found that LO inhibitors (specifi-cally of the 5-LO pathway) have chemopreventive activity inanimal lung carcinogenesis. Moodyet al. (69) and Rioux andCastonguay (70) showed that the FLAP inhibitor MK 886 (25mg/kg diet) and 5-LO inhibitor A 79175 (75 mg/kg diet)significantly reduced the multiplicity of NNK-induced tumorsin strain A/J mice; A 79175 also reduced tumor incidence. Inthe same study, aspirin (294 mg/kg diet) reduced tumor mul-tiplicity; the combination of aspirin and A 79175 (i.e., inhibit-ing both the COX and LO pathways) synergistically loweredtumor incidence and multiplicity. These results strongly suggestthat 5-LO pathway inhibitors may have chemopreventive ac-tivity in lung.

Supporting evidence links LO metabolites with lung can-cer cell growth. Studies conducted by Aviset al.(71) in severalhuman lung cancer cell lines (small cell and non-small cell)found that 5-LO is stimulated by two autocrine growth factors,GRP and IGF, both of which stimulate production of 5-HETE.5-HETE stimulated the growth of lung cancer cells, whereascells treated with 5-LO inhibitors NDGA, AA-861, and MK-886 showed decreased proliferation; the COX inhibitor aspirinhad little effect. Expression of 5-LO and FLAP mRNA by lungcancer cell lines was confirmed using reverse transcription-PCR, and the presence of 5-LO mRNA was identified in sam-ples of primary lung cancer tissue, including both small cell andnon-small cell lung carcinomas.

Also relevant to lung cancer development are studies dem-onstrating that LOs mediate oxidation of potent carcinogenssuch as benzidine,o-dianisidine, and others; this activation canbe blocked by adding the LO inhibitors NDGA and esculetin(72). Rat lung LO also oxidizes benzo(a)pyrene (73); LOs havebeen found in human lung tissue by other investigators (74, 75).Prostate Cancer. Altered eicosanoid biosynthesis in relationto prostate cancer development has been documented. Initialstudies found dramatically reduced levels of arachidonic acid;10-fold greater turnover in malignantversusbenign prostatictissue suggested a possible increase in metabolism via the LOand COX pathways in this tissue (76, 77). Linoleic acid stim-ulated cell growth in experiments conducted in human prostatecancer cells, whereas indomethacin, esculetin, and piroxicaminhibited it, substantiating the involvement of eicosanoids inprostate cancer cell proliferation (78). Although attention hasfocused on COX-derived products (79–81), particularly PGE2

(77), COX inhibitors indomethacin and aspirin failed to reducehuman prostate PC3 cell DNA synthesis, although arachidonicacid antagonist eicosatetraynoic acid did reduce synthesis (82,83), suggesting that LO products are essential in modulatingprostate DNA synthesis. However, until 5-LO products arerecovered from prostate tissue and their synthesis directlyshown to be inhibited by 5-LO inhibitory agents, other possiblemechanisms cannot be ruled out.

Additional studies by Andersonet al. (84) show reducedDNA synthesis and growth inhibition of prostate cancer cellswith specific 5-LO inhibitors (A63162; Abbott), further sup-porting involvement of the LO metabolic pathway in prostatecancer growth. Likewise, recent work by Ghosh and Myers (16)using PC-3 cells provides convincing evidence that the 5-LOmetabolic pathway stimulates prostate cancer cell growth. More

Fig. 1. LOs and arachidonic acid metabolism.

469Cancer Epidemiology, Biomarkers & Prevention



specifically, 5-HETE, particularly the 5-oxo-eicosatetraenoicform, stimulates PC-3 cell growth similarly to arachidonic acid;LTs had no effect. 5-HETEs also effectively reversed growthinhibition produced by MK-886 (Merck), a FLAP inhibitor.Both MK-886 and AA-861 effectively blocked prostate tumorproliferation induced by arachidonic acid, whereas the COXinhibitor ibuprofen and 12-LO inhibitors baicalein and BHPPwere ineffective.

Besides 5-LO products, other LO metabolites have beenimplicated in prostate tumor growth. The 12-LO metabolite12(S)-HETE plays a critical role in prostate tumor metastasisand invasion (1). In 122 matched normal and cancerous prostatetissues, 12-LO mRNA expression was confined to prostateepithelial cells and elevated in malignant cells (3). Also, ele-vated levels of 12(S)-HETE mRNA significantly correlatedwith advanced stage, poor differentiation, and invasive poten-tial of prostate cancer cells. Indeed, addition of 12(S)-HETE torat Dunning R3327 and human prostate adenocarcinoma cellssignificantly increased their cellular motility and ability toinvade basement membrane and correlated with their metastaticpotential (85–87). Additional biochemical evidence demon-strated that 12(S)-HETE stimulates secretion of cathepsin B,which is involved in tumor metastases and integrin expressionin other tumor cells (13), providing support for the importanceof LO products in the development and spread of prostate andother human cancers. More recently, Liuet al. (6) have shownthat enhancement of prostate tumor cell invasion by 12(S)-HETE involves selective activation of membrane-associatedPKCa. Furthermore, the ability of PKC inhibitor calphostin Cto block the 12-HETE-stimulated release of cathepsin B lendscredence to the observation that 12(S)-HETE acts via activationof PKC (7, 88).

Among several possible mechanisms by which LO inhi-bition may reduce prostate PC-3 cell proliferation is by alteringthe concentration of second messengers needed for continuedcell growth by shunting other arachidonic acid metabolites intoalternate pathways linked to signal transduction mechanisms(89, 90). For example, agents that inhibit 12-LO and block12-HETE production may result in widespread interference insignal transduction by preventing PKC activation (91, 92). LOinhibition may further affect the expression of proto-oncogenesor their products, including EGF, FGF, TGF-a andb, N- andK-ras, c-myc, int-2, IGF, and the retinoblastoma gene, alsodirectly or indirectly associated with normal or transformedprostate epithelial cell growth (93, 94). Whether inhibitors ofLO biosynthesis can influence expression of these factors,however, has not yet been determined.Breast. Reports published over the past two decades support agrowth-regulatory role for fatty acid metabolites and particu-larly for the arachidonic acid-derived eicosanoids in the etiol-ogy of mammary carcinogenesis (95). Feeding studies con-ducted in rodent models of mammary tumorigenesis showedthat diets rich in linoleic acid, n-6 polyunsaturated fatty acid,and arachidonic acid precursor, stimulate mammary tumorgrowth induced by either MNU or DMBA (96–99). Con-versely, dietary supplementation with n-3 polyunsaturated fattyacid, particularly fish oils, retarded mammary tumor growthand metastasis in a variety of animal tumor models (100).Studies by Abou-El-Elaet al.(101) using the DMBA mammarygland tumor model found that mammary tissue of rats fed 20%menhaden oil produced lower levels of LTB4 and PGE2 com-pared with groups receiving 20% corn oil or primrose oil,supporting the theory that PGs and LTs may be involved inmammary carcinogenesis.

Additional evidence implicating eicosanoids in humanbreast carcinogenesis stems from analysis of benign and ma-lignant breast tissue. Markedly elevated levels of both COX andLO metabolites have been documented in human breast cancertissue compared with tissue from patients with benign disease(102). Recently, Natajaranet al. (103) studied 12-LO mRNAexpression in matched, normal, nontumorous and cancerousbreast tissue from a small group of patients. In cancerous tissue,12-LO mRNA expression increased 3–30-fold compared withnormal tissue, which contained barely detectable levels. Simi-larly, greater 12-LO mRNA amounts were documented in twobreast cancer cell lines (MCF-7 and COH-BR1) compared witha noncancerous breast epithelial cell line (MCF-10).

Stimulation of breast cancer cell growth by linoleic acidhas further been substantiatedin vitro in several human breastcancer cell lines (MDA-MB-231, MDA-MB-435, and MCF-7;Refs. 104–106); the inhibitory action of other n-3 fatty acids,including docosahexaenoic acid (22:6) and eicosapentaenoicacid (20:5), have also been reported in these cells (104–106).Likewise, increased secretion of both LO and COX products,including 12- and 15-HETE and PGE2, has been documented inhuman breast cancer cells (MDA-MB-435) treated with 2.7mM

linoleic acid (104). In the same study, linoleic acid enhancedthe invasive capacity of breast cancer cells; this effect could becompletely blocked by adding esculetin (20mM), an inhibitor of5- and 12-LO, but not by adding the COX-specific inhibitorpiroxicam. Interestingly, adding 0.1mM 12-HETE mimickedthe stimulatory effect of linoleic acid on cell invasion, whereasPGE2 and 5-HETE had no effect. Collectively, these datasupport the concept that enhanced 12-HETE, not PG, is in-volved in the etiology of breast cancer cell metastasis.

Conflicting reports on the relative importance of LO andCOX in breast cancer have been published. For example, someinvestigators found that COX inhibitors such as indomethacinand flurbiprofen suppress mammary tumor development inrodent models of mammary carcinogenesis (107–109); otherstudies conducted with these agents, particularly indomethacin,yielded inconsistent results (110) or reported no correlatableeffect of COX inhibition to tumor growth (111). Consistentwith these findings,in vitro studies have not conclusivelyassociated COX inhibition with reduced mammary tumor cellgrowth (106, 112, 113). The LO inhibitors such as NDGA(0.1% diet; Ref. 114) and esculetin (0.03% diet; Ref. 115–117)inhibit rat mammary tumor development inducedin vivo byMNU or DMBA, lending credence to the hypothesis that LOproducts are important in mammary tumor development. COX-specific inhibitors produced mixed results in these studies. Forexample, the relatively selective COX-2 inhibitor nabumetone(0.03% diet) inhibited MNU-induced mammary tumor inci-dence and multiplicity (105, 117), whereas the COX-1-specificinhibitor piroxicam (0.03% diet) did not inhibit DMBA-induced tumors (116).

Additional support for the role of LO products in breastcancer development includes recently published data on theability of 12-HETE to mediate the proliferative effects of es-trogen and linoleic acid in a human breast cancer cell line (118).In this study, MCF-7 breast cancer cells, estrogen dependentand unresponsive to growth stimulation with linoleic acid, weretransfected with 12-LO cDNA and grew in the absence ofestrogen, showing reduced expression of estrogen receptormRNA and protein compared with the parental cell line. More-over, 12-LO-transfected cells grew more rapidly than the pa-rental cells (36.26 3.0 3 104 versus12.46 1.1 3 104) whencultured in media containing linoleic acid. Injection of 12-LO-transfected MCF-7 cells into mammary fat pads of ovariecto-




mized mice produced small solid tumors in 100% of estrogen-supplemented mice and 43% of unsupplemented mice.However, additional studies will be needed to explain theamplified response to estrogen in cells overexpressing 12-LOand to further determine whether 12-LO transfection increasesthe metastatic potential of these cells.

More recently, using reverse transcription-PCR and North-ern blot analysis, identification and characterization of a 15-LOidentical to the 15-LO gene product present in human pulmo-nary tissue has been reported in BT-20 human breast carcinomacells (119). BT-20 cells overexpress both the EGF receptor andthe homologouserbB-2 (neu) oncogene product, two factorsidentified as possible adverse prognostic indicators in humanbreast cancer (120, 121). Adding indomethacin to these cellshad no effect on EGF and TGF-a-stimulated DNA synthesis,nor did it block the LO-mediated metabolism of linoleic acidinto 13-HODE. NDGA, on the contrary, inhibited production of13-HODE and attenuated TGF-a-induced DNA synthesis, sup-porting a role for LO metabolites in regulating the EGF receptorsignaling pathway. Thus, increased production of 13-HODE asa result of high dietary levels of linoleic acid may up-regulateand amplify the EGF mitogenic signaling pathway, resulting inenhanced cell growth in cancerous breast tissue. On the otherhand, reducing 13-HODE via LO inhibition may potentiallyinhibit the mitogenic response and block further proliferation ofbreast cancer tissue.

Otherin vitro studies postulated that LO metabolism maybe involved in modulating tumor cell adhesion. In one studyconducted in the human breast carcinoma cell line MDA-MB-435, exogenous NDGA dramatically inhibited adhesion to col-lagen IV induced by either A23187 or arachidonic acid,whereas indomethacin had no effect, implying that COX is notinvolved in this process (122). Consistent with these findings,previous reports showed that linoleic acid potentiates the met-astatic potential of this cell line bothin vitro and in vivo (123,124) and also stimulates cell adhesion to fibronectin in anotherhuman breast cancer cell line (125).Colon. Besides overwhelming data linking PGs to the etiologyof colon carcinogenesis (126), several recent lines of evidencesuggest that LO products may also be involved in this process.Specifically, in vitro studies conducted in two colon cancercells lines (HT-29 and HCT-15) reported time- and dose-de-pendent stimulation of cell proliferation by LTB4 and 12(R)-HETE, a P450-derived product (127). In the same study,SC41930, a competitive LTB4 antagonist, inhibited LTB4-in-duced growth stimulation in HT-29 cells. Similar inhibitionoccurred in murine colon adenocarcinoma cell lines MAC16,MAC13, and MAC26 treated with other 5-LO inhibitors, in-cluding BWA4C and BWB70C at micromolar concentrations(IC50, ,10 mM), whereas Zileuton (IC50, 40 mM) was lesseffective (128). The same study further analyzedin vivo activ-ities of these agents in male NMRI mice transplanted withfragments of MAC26 or MAC16 colon tumors. At 25 mg/kgbody weight, BWA4C was the most effective inhibitor, signif-icantly decreasing both growth rate and tumor volume after8–13 days of treatment.

Additional evidence supporting LT biosynthesis in coloncancer development stems from biochemical and genetic stud-ies. The ability of colonic epithelial cell lines to synthesizesome LTs, including LTB4 and LTA4, has been confirmed inhuman HT-29 (129, 130) and CaCo-2 cells (131) and in ratcolonic crypt epithelial cells (132). Northern blot analysis oftotal RNA revealed low levels of mRNA transcripts for both5-LO and FLAP (129), suggesting that intestinal cells have a

limited capacity to synthesize LTs. The low recovery of LOenzyme transcripts is consistent with the theory that LTs serveas intracellular messengers rather than inflammatory mediatorsin this tissue. More recently, platelet-type 12-LO mRNA hasbeen recovered from a human colon carcinoma cell line (2);treatment of these cells with 12(S)-HETE resulted in up-regu-lation of 12-LO mRNA and protein (133). Collectively, thesedata strongly suggest that LO metabolism may play a role incolon tumor proliferation.Skin. Considerable evidence suggests that LOs are involved inepidermal tumor development. Compared with normal epider-mis, large quantities of 12(S)-HETE (50–60-fold greater) werefound in papillomas and carcinomas induced by DMBA andTPA in a mouse skin tumor model (134). In the same study,12-LO enzyme activity was elevated 6-fold in papillomas and3-fold in carcinomas compared with normal tissue. Moreover,expression of platelet-type 12-LO has been confirmed in bothnormal human epidermis (135) and human epidermoid A431carcinoma cells (66). 12(S)-HETE overproduction in papillo-mas may be a mechanism for progression to malignant carci-noma. Recent studies found that EGF and TPA up-regulateexpression of 12-LO mRNA in the human A431 cell line (35,66, 136). Overexpression of Ha-ras in these cells increased thetranscription of 12-LO in a dose- and time-dependent mannerthat correlated to the cellular expression of ras protein (137).Although this study provides evidence thatras can activate12-LO gene transcription directly, it did not determine whetheragents that inhibit 12-LO would be effective in preventingras-mediated stimulation of intracellular signaling pathways.Other Sites. In squamous epithelial carcinomas of the headand neck, 12- and 15-HETE are major arachidonic acid metab-olites (138). Also, 12(S)-HETE is the predominant metabolicproduct of metastatic B16 melanoma cells (139). Additionally,excess LT production, specifically LTC4, has been documentedin cells from patients with both acute and chronic leukemias(140–142). Adding 5-LO inhibitors SC 41661A (Searle) andA63162 (Abbott) to these cells reduced DNA labeling anddecreased cell numbers within 72 h (140, 143). Likewise,growth inhibition with other LO inhibitors, including piriprost,NDGA, and BW755C, has been demonstrated in several ma-lignant human hematopoietic cell lines; the COX inhibitorindomethacin lacked a suppressive effect (47, 144). These dataimply that LO products are essential for thein vitro growth ofmalignant hematopoietic cells.

Pharmacological AgentsAs suggested in the discussions above, two general classes ofagents inhibit LO pathways. First are the (usually) specificinhibitors of 5-LO and FLAP, and (usually) nonspecific inhib-itors of 12-LO; second are LT receptor antagonists. Althoughthe latter class of agents has high potential to treat asthma andother diseases where drug effects are clearly mediated by LTreceptors, inhibitors of specific enzymes in the LO pathwaymay be better candidates for chemopreventive intervention.Unlike receptor antagonism, inhibition of these enzymes resultsdirectly in reducing the production of fatty acid metaboliteswith concomitant damping of the associated inflammatory,proliferative and metastatic activities that contribute to carci-nogenesis. In the following paragraphs, both enzyme and re-ceptor inhibitors are described. However, because they arecommercially available and, hence, well characterized, andbecause they have aerosol formulations and possible antipro-liferative activity, the receptor antagonists may also have che-




Table 2 LO inhibitory agents

Agent (developer) TargetStatus

(indication)LO inhibitory

activity (in vitro, IC50)

LO and otherinhibitory activities

(in vivo, ED50)

Antiproliferative activity(in vitro, IC50)

Zileuton Leutrol Zyflo(Abbott)

5-LO FDA approved 12/96,launched in the UnitedStates January 1997(asthma)

RBL-1 (0.5 mM) and ratPMNLa (0.3 mM) 5-HETEsynthesis (146)

Human whole blood (0.9mM)and PMNL (0.4mM) LTB4

(146)

Rat pleural effusion LTB4inhibition 1–3 mg/kg p.o.(228); 70% decrease inexvivo whole-blood LTB4

synthesis in humans given600 mg q.i.d. p.o.3 14days (229)

Murine colonadenocarcinoma cells(43–58mM) (128)

Plasma half-life 2.5–3 h p.o.(230)

ABT-761 (Abbott) 5-LO12-LO

Phase III (asthma) LTB4 inhibition: RBL-1, andhuman PMNL (23 nM),human whole blood (150nM) (149)

A single 200-mg p.o. dosegave 95% inhibition ofLTB4 in humanex vivoblood for up to 18 h (149)

Not reported

Human whole blood 12-HETE synthesis (11mM)(149)

Human plasma half-life 200mg p.o. 15 (149)

AA-861 (Takeda) 5-LO12-LO

Discontinued Phase II(asthma, allergy)

Guinea pig peritoneal PMNL5-LO (0.8 mM) (231);Mouse epidermal 12-LO(1.9 mM) (150, 151)

30 mg/kg i.p. inhibited TPA-induced ODC activity inmouse liver, spleen, andkidney (152); TPA-induced neutrophilinfiltration blocked at 10mmol/mouse (232)

Human leukemia cells (6–20mM) (153)

Lung cancer cells stimulatedwith IGF and GRP (5–10mM) (71)

ZD2138 (Zeneca) 5-LO Discontinued Phase II(asthma)

LTB4 inhibition in human(0.024mM), rat (0.033mM), and dog (0.02mM)blood (233)

Rat inflammatory exudatemodel (0.3 mg/kg p.o.)(233)

Not reported

At 350 mg p.o., completeinhibition of LTB4 in exvivo human whole blood;human plasma half-life12–16 h (234)

MK-886 (Merck) FLAP Discontinued Phase I(asthma)

Human and rat neutrophil LTbiosynthesis (3–5 nM),intact human PMNs(2.5 nM) (157)

Rat pleurisy model (0.2–2.3mg/kg p.o.), rat pawedema (0.8 mg/kg), ratbronchospasm assay(0.036 mg/kg) (157); 750mg p.o. produced 50%inhibition in ex vivohuman whole-blood LTB4(159)

DNA synthesis inhibition inacute myelogenousleukemia cells at 100 nM(235)

Growth inhibition of chronicmyelogenous leukemia(10–20mM) (141) andlung cancer cells (5–10mM) (71)

MK-0591 (Merck) FLAP Discontinued Phase I(asthma, ulcerative colitis)

LT synthesis in humanPMNLs (3 nM) and ratneutrophils (6 nM);leukocyte FLAP bindingassay (23 nM) (236)

750 mg p.o. daily reducedbronchoconstriction anddecreased LT synthesis by98% in humanex vivostimulated whole blood(237)

Not reported

BAY-X1005 (Bayer) FLAP Discontinued Phase II(asthma, cardiac failure)

Human (0.22mM) and ratPMNLs (0.026mM);mouse macrophage (0.021mM) LTB4 synthesis (162)

Arachidonic ear edema (49mg/mouse) (238)

Not reported

LTB4 inhibition in rat ex vivowhole blood (11.8 mg/kgp.o.) (162)

Oral half-life at 50–750 mg4–8 h (239)

SC 41930 (Searle) LTB4 receptor Discontinued Phase II(asthma, colitis)

Human PMN LTB4-inducedneutrophil degranulation(1080 nM), human PMNchemotaxis assay (832 nM)(168)

PMA-induced ear edema inrodents (4.2mM/ear) (168)

Inhibits fMLP-inducedsuperoxide release (167)

Not reported

SC 53228 (Searle) LTB4 receptor Phase I (asthma, ulcerativecolitis)

Human PMN LTB4 receptorbinding (1.3 nM), LTB4-induced neutrophilchemotaxis (32 nM) (168)

LTB4-induced neutrophilchemotaxis in guinea pigskin (70mg/kg bodyweight); guinea pig p.o.half-life 9 h (240)

Not reported




mopreventive potential, particularly in lung, where topical ap-plication of such formulations is feasible.

Initial 5-LO inhibitors were nonspecific antioxidants thatlacked oral bioavailability, produced adverse physiological ef-fects, or interfered with biological processes. Early 5-LO in-hibitory compounds, such as phenidone, and various analoguesincluding BW755C, A53612, and ICI 207968, induced methe-moglobinemia, precluding their further clinical development(145). Identifying natural products with LO inhibitory activity,such as caffeic acid, NDGA, and vignafuran, inspired pharma-ceutical researchers to develop other compounds and analoguesof greater potency and enzyme specificity. These efforts led todevelopment of specific 5-LO inhibitors, FLAP inhibitors, pep-tidyl LT receptor antagonists, and LTB4 receptor antagonists(Table 2 and Figs. 1-5). Experimental agents demonstrating12-LO inhibitory activity (Table 3) are shown in Fig. 6. Thefollowing discussion addresses each class of compounds andincludes some agents that have undergone clinical evaluation.

5-LO InhibitorsFirst Generation N-Hydroxyurea Derivatives. Zileuton[Leutrol, N-(1-benzo(b)-thien-2yl) ethyl-N-hydroxyurea], aspecific 5-LO inhibitor of theN-hydroxyurea series developedby Abbott, represents the first p.o. active LT inhibitor to showclinical efficacy in humans (145). Zileuton reportedly inhibits5-LO via iron chelation but is devoid of 12- and 15-LO inhib-itory activity (146). After extensive clinical evaluation, Zileu-ton received FDA approval in December 1996 for treatingbronchial asthma in patients 12 years and older and waslaunched in the United States in January 1997. At oral doses of600 mg four times daily, Zileuton produces moderate airwayfunction improvement in asthmatics within 30 min. An ex-tended release formulation (Zyflo) with reduced twice-dailydosing frequency is also available. In addition to asthma, Zileu-ton has also been evaluated to treat ulcerative colitis. However,work on this indication was halted in 1993 after a Phase IIIclinical trial that found that Zileuton, although effective, had nosignificant advantage over other conventional therapies.

The antiproliferative properties of Zileuton have not yetbeen investigated extensively. One study compared the growth-suppressive effects of Zileuton and two hydroxamate inhibitors,BWA4C and BWB70C (Wellcome) in three different murinecolon adenocarcinoma cell lines (MAC16, MAC13, and

MAC26; Ref. 128). All three compounds effectively reducedcell proliferation in all cell lines tested; Zileuton showed 10-fold higher IC50s (43–58mM) than the other compounds (2–5mM). Because of toxicity demonstrated by both BWA4C andBWB70, further clinical development of these agents has beenterminated (147). Additional testing is needed to fully evaluatethe antiproliferative action of Zileuton and to determine itspotential as a chemopreventive agent.Second GenerationN-Hydroxyurea Compounds. The pri-mary drawbacks of Zileuton are its relatively short activityduration and high effective dose, both likely resulting fromextensive glucuronidation and subsequent rapid excretion. Thesearch for similar compounds with greater potency and reducedglucuronidation rate led to the discovery of a number of struc-turally different moieties, including the (R)-1-methoxypropynylseries represented by Abbott’s A78773 and A79175 (ABT-175). A78773 proved to be more potent than Zileuton bothinvitro andin vivo (148). A78773 was;30-fold more potent thanZileuton in the ionophore-stimulated neutrophil assay.Ex vivoevaluation of A78773 after a single oral dose of 0.5 mg/kg bodyweight revealed complete inhibition of LTB4 production (95%)for up to 5 h and 70% inhibition for 12 h. At the same doselevel, Zileuton showed 70% inhibition for 2 h but steadilydeclined thereafter (148). The antiproliferative activity ofA78773 has not been determined.

A79175, the (R1) racemate of A78773, exhibited similarpotency to A78773 but showed greater resistance to glucu-ronidation. A79175 entered Phase I clinical trials but wassuspended after the discovery of synthetic intermediates of the((4-fluoro-phenyl)methyl)-2-thienyl series represented byA85761 (ABT-761), which showed greater stability. In a PhaseI study of ABT-761, a single oral dose of 200 mg produced aplasma half-life of 15 h with plasma levels of 1mg/ml for up to72 h after dosing (149). The extended duration of action dem-onstrated by ABT-761 may be therapeutically beneficial inchemoprevention trials where sustained 5-LO inhibition is de-sirable.Redox Inhibitors. One of the most widely studied redox in-hibitors is docebenone or 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA-861), a lipophilic qui-none structurally resembling coenzyme Q developed by TakedaChemical Industries, Ltd. (Osaka, Japan). AA-861 is a potentcompetitive inhibitor of 5-LO but has no effect on either 12-LO

Table 2 Continued

Agent (developer) TargetStatus

(indication)LO inhibitory

activity (in vitro, IC50)

LO and otherinhibitory activities

(in vivo, ED50)

Antiproliferative activity(in vitro, IC50)

Ultair Pranlukast ONO-1078(SmithKline Beecham/OnoPharmaceuticals)

LTD4 receptor Approved and launched inJapan 1995 (asthma,allergy, pruritus); Phase III(asthma) and Phase I(pediatric asthma) inUnited Kingdom andUnited States

26-fold shift in LTD4-inducedbronchoconstrictionresponse curve at 450 mgp.o. twice daily (241)

Not reported

Significant improvement inlung function at 225–450mg twice daily in chronicasthmatics (241, 242)

Zafirlukast Accolate(Zeneca)

LTD4 receptor Approved for marketingSeptember 1996 in UnitedStates (asthma)

LTD4 binding (K1 5 0.3 nM)(170)

A single 40-mg oral dose inhealth volunteers produceda 117-fold shift in theLTD4-inducedbronchoconstrictionresponse curve (243)

Not reported

a PMNL, polymorphonuclear lymphocyte.




or COX at concentrations,10 mM. In epidermal homogenatesof CD-1 mice, AA-861 is a potent inhibitor of epidermal 12-LO(IC50 1.9 mM; Refs. 150 and 151).

In addition to its anti-inflammatory properties, AA-861(15 mM/mouse) strongly suppressed skin tumor formation in-duced by DMBA and TPA (10 nmol/mouse) and produceddose-dependent inhibition of TPA-stimulated ODC activity inthe mouse at concentrations of 1–30mM (151). Additionalinvivo studies further showed that at 30 mg/kg body weightadministered i.p., AA-861 prevented TPA-induced increase inODC activity in liver, spleen, and kidneys of CD-1 mice (152).

Tsukadaet al. (153) first reported the antiproliferativeactivity of AA-861 in a study of three human leukemia celllines, K562, Molt4B, and HL60, and the human cervical cellline HeLa. In this study, AA-861 (6.7 and 20mM) markedlyreduced the growth of leukemia cells but had no effect oncervical HeLa cells. Similarly, at 5–50mM, AA-861 produceddose-dependent growth inhibition in murine mastocytoma cells(P815; Ref. 154). More recently, AA-861 at concentrations of;5–10mM significantly reduced the growth and proliferation ofhuman small cell lung cancer (NCI-H209, H345, H82, andN417) and non-small cell lung cancer (NCI-H1155, H23) celllines stimulated by either of two autocrine growth factors, IGFand GRP (71). Profiles of arachidonic acid metabolism inAA-861-treated cells showed consistent inhibition of 5-HETE,LTD4, and arachidonic acid metabolism. Another study in-duced apoptosis in rat Walker (W256) carcinosarcoma cellswith AA-861 at concentrations of$20 mM (155).

Non-Redox Inhibitors. ZD2138 by Zeneca, a selective, p.o.-active 5-LO inhibitor of the methoxytetrahydropyran series, isdevoid of redox and iron ligand-binding properties (156). De-spite its promising anti-inflammatory profile, Phase II clinicaltrials carried out in asthmatics had mixed results (145), haltingfurther clinical development of this compound. WhetherZD2138 exerts antiproliferative activity remains to be deter-mined.FLAP Inhibitors, Indole Series. Extensive screening of in-dole compounds derived from COX inhibitors indomethacinand sulindac led to development of MK-886 by Merck (157),the first FLAP inhibitor to reach clinical evaluation. MK-886 isbelieved to work by binding to an arachidonic acid binding siteon FLAP, facilitating the transfer of the substrate to 5-LO(158). Asthmatics administered 750 mg of MK-886 p.o.showed only 50% inhibition of LTB4 in ex vivo-stimulatedwhole blood and in urinary LTE4 levels (159). On the basis ofthese data, further clinical development of MK-886 was dis-continued. However, antiproliferative effects of MK-886 wereobserved recently at micromolar concentrations in malignantcells from patients with chronic myelogenous leukemia (141)and in human lung cancer cells (71). On the basis of thesepreliminary findings, MK-886 may have some therapeutic po-tential as a chemopreventive agent.

MK0591 represents a novel 2-indolealkanoic acid deriv-ative and second-generation FLAP inhibitor developed byMerck (160). Like MK-886, MK0591 blocks 5-LO activity bybinding to FLAP, thereby preventing 5-LO translocation andactivation. Despite its promising biochemical profile, develop-ment of MK0591 was discontinued after results from furtherclinical testing were below anticipated values.FLAP Inhibitors, Quinoline Series. Optimization of the2-quinolylmethyloxy phenyl residue of Revlon’s REV 5901 ledto BAY-X1005, a potent, p.o. active inhibitor of 5-LO devel-oped by Bayer AG to treat asthma (161, 162). BAY-X1005reportedly lacks 12-LO or COX inhibitory activity and is de-void of antioxidant activity (163). The binding of BAY-X1005to FLAP has been reported to directly correlate with the degreeof LTB4 inhibition (164). Development of BAY-X1005 for

Fig. 2. 5-LO inhibitors.

Fig. 3. FLAP inhibitors.




treating asthma was discontinued after a Phase III clinicalevaluation. The antiproliferative capacity of BAY-X1005 hasnot been reported in the literature.LTB 4 Receptor Antagonists.SC 41930, a potent first gener-ation LTB4 receptor antagonist developed by Searle (Mon-santo; Refs. 165 and 166) has demonstrated potency in a varietyof inflammatory models. However, the discovery that SC41930 inhibits f-MLP-induced superoxide release (167)prompted further research to develop agents with greater po-tency and selectivity.

Second-generation agents derived from structural ana-logues of SC 41930 include monomethyl amide SC 53228 (168,169) and thiazole analogue SC 50605 (7-(3-(2-(cyclopropyl-methyl)-3-methoxy-4-(thiazol-4-yl)phenoxy)propoxy)-3,4-di-hydro-8-propyl-2H-1-benzopyran-2-carboxylic acid) (167).Potent antagonists of LTB4 receptors both demonstrate sub-stantially improved pharmacological profiles compared withSC 41930. SC 50605 and SC 53228 have been selected forfurther clinical evaluation.LTD 4 Receptor Antagonists.Ultair (Pranlukast, ONO-1078,SB205312) or N-(4-oxo-2-(1H-tetrazol-5-yl)-4H-1-benzopy-ran-8-yl)-4-(4-phenylbutoxy)-benzamide, licensed from OnoPharmaceuticals by Smithkline Beecham, is the first LTD4

antagonist to be introduced in the world, having been approvedto treat asthma in Japan in 1995; it is now in Phase III clinicaltrials in the United Kingdom and United States. Whether thispharmacological agent or other LTD4 antagonists exert anti-proliferative activity has not been determined.

Zafirlukast or cyclopentyl 3-(2-methoxy-4-((o-tolylsulfo-nyl)carbamoyl)benzyl)-1-methylindole-5-carbamate (Accolate,

ICI-204,219; Zeneca), an LTD4 receptor antagonist, wascleared in September 1996 for marketing as an asthma treat-ment in the United States. Accolate has demonstrated potentactivity both in vitro and in vivo (170). Upon receiving FDAapproval, the Pulmonary-Allergy Drug Advisory Committeehas declared Accolate safe and effective in patients older than12 years of age with mild to moderate asthma (171). Thechemopreventive efficacy of this agent, however, has not beeninvestigated.Esculetin and Other Experimental 12-LO Inhibitors (Non-specific). Esculetin, a coumarin derivative extracted from thebark of Aesculus hippocastanum, is a well-known competitiveinhibitor of both the LO and COX metabolic pathways (172–175). At concentrations of 0.1–1mM, esculetin exerts LO in-hibitory activity; levels.10 mM are required for COX inhibi-tion (172, 175). Acute toxicity testing of esculetin in the mouseproduced an LD50 of 1450 mg/kg body weight i.p. and 2000mg/kg when given p.o. (176).

The antiproliferative action of esculetin has been demon-strated in severalin vitro models. At concentrations of 3–30mg/ml, it significantly reduced LTB4 secretion and producedgrowth suppression in the MDA-MB-231 human breast cancercell line (177). Likewise, esculetin at 20mM completely sup-pressed production of 12-HETE in estrogen-independent hu-man MDA-MB-435 breast cancer cells stimulated with linoleicacid (104). In the same study, 2 or 20mM of esculetin com-pletely blocked the invasiveness of cells stimulated by linoleicacid while completely suppressing type IV collagenase (met-alloproteinase-9) mRNA expression. Similar growth-suppres-sive effects of esculetin have been reported in human T-lymph-oid leukemia cells (IC50, 3.6 6 0.3 mM; Ref. 178) and invascular smooth muscle cells (IC50, 10.96 2.8mM) stimulatedwith 5% FCS (179).

In vivo studies have further shown that esculetin signifi-cantly inhibits the development of mammary tumors induced

Fig. 4. LTB4 receptor antagonists.

Fig. 5. Peptidyl LT antagonists.




by DMBA in female Sprague Dawley rats fed either a high-fat(20% soybean oil) or low-fat (0.5% soybean oil) diet (115,116). Rats administered diets containing 0.03% esculetinshowed significant reduction in mammary tumor incidence,tumor growth, and cell kinetics in both the high- and low-fatdietary groups. More recently, esculetin has been effective inreducing the invasive and metastatic activity of malignant tu-mor cells (180). Murine melanoma cells pretreated with 50mM

esculetin and injected into syngeneic mice produced signifi-cantly lower numbers of melanoma colonies on the lung surfaceand fewer tumor metastases. Thus, esculetin may be an effec-tive chemopreventive agent on the basis of these data; however,additional studies will be needed to assess its efficacy in othertumor models.

Baicalein, a low molecular weight flavonoid isolated fromScutellaria baicalensisGeorgy roots, is a key component ofJapanese herbal medicine Sho-saiko-to, commonly used to treatchronic liver diseases in Japan. Early studies conducted in ratplatelets showed that nanomolar concentrations of baicaleinexert potent 12-LO inhibitory activity, whereas much higherlevels are required for COX inhibition (181). In rat PMNs,baicalein also reportedly blocks 5-LO and production of LTC4

(182).The antiproliferative effects of baicalein have been re-

ported in severalin vitro models. Mootoet al. (183) showedthat baicalein (IC50, 50 mg/ml) reduced DNA synthesis andinhibited growth in two human hepatoma cell lines (phospho-lipase C/PRF/5 and Hep-G2). Similar growth-suppressive ef-fects of baicalein have been reported by other investigators invarious hepatocellular cancer cell lines, particularly HuH-7cells (184–186). Likewise, treating human breast cancer cells(MCF-7) with baicalen at microgram levels produced potentantiproliferative activity (IC50, 5.3mg/ml; Ref. 187). In humanT-lymphoid leukemia cells (CEM) stimulated with 10% FCS,baicalein dose dependently reduced cell proliferation (IC50,4.76 0.5mM), exhibiting maximal inhibition (91.56 1.4%) at1024

M (178). In the same study, baicalein reduced the expres-sion of PDGF-A mRNA, and to a lesser degree also affectedTGF-b1 mRNA expression, suggesting that baicalein may exertits antiproliferative action via inhibition of growth factor-me-diated signaling pathways. Additional studies found that 5–100mM concentrations of baicalein in rat Walker (W256) carcino-sarcoma cells induced apoptosis in a dose-dependent manner(155). Whether baicalein exerts similar antiproliferative activity

in vivo, however, remains to be determined. Nevertheless, theseinitial data suggest that baicalein may be an effective chemo-preventive agent.

Another select 12-LO inhibitor is BHPP, one of a series ofhydroxamic acids originally developed by Rorer that was foundto exert 5-LO inhibitory activity in rat leukocytes (188). Ad-ditional studies demonstrated 12-LO inhibitory activity inLewis lung carcinoma cells and porcine leukocytes (189) and inmurine B16a melanoma cells (7).

The antiproliferative action of BHPP has recently been

Fig. 6. 12-LO inhibitors.

Table 3 Experimental compounds demonstrating 12-LO inhibitory activity

Compound Activity (in vitro) Antiproliferative activity Chemopreventive activities

Esculetin (coumarin derivative) Inhibits 5-LO at 0.1–1mM, COX at 10mM

(172, 175);mastocytoma 12-LOIC50 5 2.5 mM (173)

Decreased LTB4 and produced growth suppressionin human T-cell leukemia (177), vascularsmooth muscle (179), and breast cancer cells(177) at 3–30mM

0.03% in diet reduced incidence of DMBA-inducedmammary tumors in SD rats (115, 116)

Pretreatment with 50mM reduced metastasis andinvasiveness of melanoma cells injected intosyngeneic mice (180)Antiinflammatory and analgesic activity reported

(176)

Baicalein (flavonoid) Rat platelet 12-LO IC50

5 0.12 mM (181), ratPMN 5-LO (IC50,9.5 mM) (182)

Reduced DNA synthesis and produced growthinhibition in human liver cells (183), breastcancer (187), and T-lymphoid leukemia (178)

Induced dose-dependent apoptosisin vitro in ratcarcinosarcoma cells at 5–100mM (155)

Reduced PDGF-A and TGF-a mRNA in leukemiacells (178)Antiinflammatory activity reported in arthritis

(182) and other models (244)

BHPP (hydroxamic acid) Inhibits porcine leukocyteand Lewis lungcarcinoma 12-LO atmicromolar levels(189) and rat leukocyte5-LO (188)

Growth inhibition in rat Walker carcinosarcomacells at 0.01–100mM (190)

40–50% reduction in lung colony formation inmice given 50 mg/kg body weight p.o. or i.p.and injected with metastatic HM340 cells (2)




reported in rat Walker (W256) carcinosarcoma cells. At con-centrations of 0.01–100mM, BHPP demonstrated dose-depen-dent growth inhibition; at 10mM, it produced DNA fragmen-tation and induced apoptosis in treated cells (190). BHPP hasbeen studiedin vitro to assess the role of 12-LO and 12(S)-HETE production in tumor cell matrix interactions and analyzeintegrin aIIbb3 expression andin vivo in an experimentalmodel of tumor metastasis (2). In these studies, murine B16aHM340 cells of high metastatic potential were exposed toincreasing concentrations of BHPP (1025–1028

M). Cellstreated with BHPP showed decreased adhesion to fibronectinand subendothelial matrix and reduced surface expression ofaIIbb3. Oral and i.p. administration of BHPP (50 mg/kg bodyweight) 30 min before tail vein injection of HM340 cellsproduced a 40–50% reduction in lung colony formation insyngeneic C57BL/6J mice. Additional testing will be needed toverify the potency of BHPP in models of chemoprevention.

Representative LT inhibitory agents that have been clini-cally evaluated but not specifically addressed in this report arelisted in Table 4. These include WY50295 (Wyeth-Ayerst),Montelukast or MK-476 (Merck), LY29311 (Lilly), CGS-25019C and Iralukast or CGP-45715A (Ciba Geigy/Novartis),ONO-4057 (Ono Pharmaceuticals), SB-210993 and SB-209247(SmithKline Beecham), SC-53228 and SC-52798 (Searle), andBAY-X7195 (Bayer). Because these agents have also demon-strated LO inhibitory potency, their potential as chemopreven-tive agents cannot be ruled out. However, additional studieswill be needed to verify the therapeutic utility of LO inhibitorsin chemoprevention.

Assessing LT Inhibitory ActivityTo assess potency and enzyme specificity, pharmacologicalprofiles of each agent are established based on screening dataobtained from both cell-free assays andin vitro model systems.For this purpose, each compound is subjected to a series ofenzymatic screens to determine 5-, 12-, and 15-LO inhibitoryactivities. 5-LO measurement is typically conducted in celllysates from rat (RBL-1) cells. According to this protocol,RBL-1 cells are harvested and lysed by sonication and centri-fuged, and the supernatant fraction was used as a source of theenzyme (146). Activity (5-HETE production) is then measuredby either RIA or enzyme immunoassay. Alternately, activitycan be determined by using [14C]arachidonic acid as substrate,followed by extraction and separation via TLC or reverse-phasehigh-performance liquid chromatography with on-line radiation

detection. In a similar fashion, 12-LO can be assessed in lysatesof human platelets or in partially purifed preparations availablethrough commercial sources. Likewise, purified forms of rabbitreticulocyte or soybean 15-LO can be obtained through localsuppliers.

In addition to these sources, methods have been developedto purify 5-LO from human leukocytes using ATP-agaroseaffinity chromatography (191–193). According to this protocol,;500 mg of protein can be recovered from 83 108 cells.Additionally, glycogen-induced rat peritoneal PMNs are analternate source of 5-LO when cells are harvested and lysed,then centrifuged. The resulting supernatant is used to measure5-LO activity as above. Using 105,0003 g supernatant frac-tion, the major arachidonic acid metabolites produced are LTB4

and 5-HETE.Once activity profiles are determined in cell-free assays,

each agent is further evaluated in whole-cell models, furtherverifying LO biosynthesis and establishing the inhibitory effi-cacy of each agent. Examples of intact cell models used inassessing eicosanoid biosynthesis include human whole bloodor PMNs, washed platelets (12-LO), and rat peritoneal leuko-cytes. In these experiments, cells are preincubated in the pres-ence and absence of varying concentrations of inhibitors, thenstimulated with ionophore. LO metabolites are extracted andmeasured using either enzyme immunoassay or RIA or prela-beled with radiolabeled arachidonic acid before separation byTLC or high-performance liquid chromatography.

Cell Proliferation and DNA Synthesis AssaysOnce inhibitory potency has been established in whole-cellmodels, each agent is further subjected to a series of antipro-liferative screens using established cell culture cancer assaysystems. These assays should include representative human celllines for breast, colon, prostate, and lung cancer. Experimentsdetermine LT or HETE-mediated growth stimulation and con-centration-dependent inhibition of cell proliferation and DNAsynthesis for each agent. Cell proliferation can be measured bythe colorimetric method of Mosmann (194) using tetrazoliumsalt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide. Briefly, cells are plated at a uniform density and allowedto incubate for a given time period (usually several days) in thepresence or absence of inhibitors, then processed according to3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideprotocol. For DNA synthesis analysis, cells are plated at auniform density, then exposed to [3H]thymidine and allowed to

Table 4 Representative Leukotriene Inhibitors/Antagonists Not Included in Text

Agent (developer) Target Indication Clinical status

CGS-25019C (Novartis/Ciba-Geigy) LTB4 antagonist Asthma, bronchitis, rheumatoid arthritis Phase IIONO-4057 (Ono Pharmaceutical) LTB4 antagonist Psoriasis, ulcerative colitis, Behcet’s disease,

inflammatory bowel diseasePhase II

SB-201993 (SmithKline Beecham) LTB4 antagonist Inflammation, psoriasis Discontinued Phase IISB-209247 (SmithKline Beecham) LTB4 antagonist Psoriasis, eczema Phase IISC 52798 (Searle) LTB4 antagonist Inflammation, psoriasis, ulcerative colitis DiscontinuedVML-295/LY29311 (Vanguard/Eli Lilly) LTB4 antagonist Asthma, inflammation, inflammatory bowel

disease, psoriasis, rheumatoid arthritis,ulcerative colitis

Phase II

WY-50295 (Wyeth-Ayerst) 5-LO/LT antagonist Arthritis, asthma, psoriasis, inflammation Discontinued Phase IIMontelukast/MK-476 (Merck Frosst) LTD4 antagonist Asthma (patients$6 years of age) New Drug Application filed first quarter

1997, approved in Finland September1997

BAY-X7195 (Bayer) LTD4 antagonist Asthma Phase IIIralukast/CGP-45715A (Novartis/Ciba Geigy) LTD4, LTE4 antagonist Asthma Phase II




incubate for 1–4 h in the presence and absence of inhibitors.After incubation, the cells are washed, and residual label isassessed by scintillation counting. Cell viability is determinedin the presence of inhibitors to rule out any agent-induced toxiceffects by either trypan blue exclusion methodology or by thecolorimetric method of Mosmann (194) described above.In Vivo Testing. The chemopreventive efficacy of select in-hibitory agents can be further tested in well-establishedin vivocarcinogen models (195). These include MNU- and DMBA-induced mammary carcinogenesis assays conducted in femaleSprague Dawley rats (196–198). Both models require a singleadministration of the carcinogen; mammary tumors are pro-duced within 65–80 days. The strain A/J mouse lung adenomamodel is generally used to evaluate the chemopreventive ac-tivity of agents against lung cancer (199). It may be advanta-geous to administer the LO inhibitors by aerosol inhalation toavert systemic toxicity (200).

Potential inhibitors of colon carcinogenesis have beenassessed using rat (F344) and mouse (CF1) species (201, 202).For example, according to established protocols, a single doseof azoxymethane administered s.c. to male F344 rats causescolon adenoma and adenocarcinoma formation in about 70% oftreated animals by 40 weeks (202). Short-term colon tumormodels include measurement of ACF in portions of rat colonstained with methylene blue (203, 204). ACF, thought to bepreneoplastic lesions that eventually give rise to colon tumors,are used frequently as intermediate biomarkers of colon cancer(205). Test agents are generally administered p.o., either before,during, or after a single or multiple dose of azoxymethane.Efficacy is evaluated as a reduction in the number of ACF afterhistological analysis.

Several models have been described for evaluating effectson prostate carcinogenesis. These include the Noble rat modelin which male rats are treated with testosterone and estradiol(206, 207), and a second, more recent model developed byBosland, whereby Wistar rats are given cyproterone acetate,MNU, and testosterone (208, 209). Evaluation of skin tumorinhibition will be conducted according to a two-stage skintumorigenesis protocol (210) in which DMBA and TPA induceskin papillomas and squamous cell carcinomas (211, 212).Before initiation with DMBA or after promotion with TPA, testagents are applied topically to the back of SENCAR or CD-1mice, which are highly susceptible to skin tumor induction.

Once the most appropriate studies are completed, activityprofiles of the agents will be compared to identify compoundssuitable for chemoprevention trials in human subjects. Note thatsome test compounds have already demonstrated activity inlung and breast. Agents that can be obtained through commer-cial or other reliable sources and for which pharmacologicaland preclinical safety data already exist will be recommendedinitially for further safety evaluation and clinical testing.

Because LO inhibitors are antiasthmatics, conducting fu-ture chemoprevention studies and applications in the lung has ahigh priority. Although approved pharmaceuticals in this classare bioavailable p.o., as noted above, it could be beneficial toexplore the use of inhalant formulations, which would deliveragent locally to the lung, potentially reducing toxicity andallowing higher, more efficacious doses. The prostate is also acancer target of high interest, because LO inhibitors demon-strate a high rate of arachidonic acid metabolism and antipro-liferative activity in prostate cancer cells. If these agents proveto be low-toxicity inhibitors of oxidative fatty acid metabolism,the breast will be a potential target for further study.

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1999;8:467-483. Cancer Epidemiol Biomarkers Prev Vernon E. Steele, Cathy A. Holmes, Ernest T. Hawk, et al. ChemopreventivesLipoxygenase Inhibitors as Potential Cancer

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