12-Lipoxygenases and 12(S)-HETE: role in cancer metastasis

Cancer and Metastasis' Reviews 13: 365-396, 1994. © 1994 Kluwer Academic Publishers. Printed in the Netherlands.

12-Lipoxygenases and 12(S)-HETE: role in cancer metastasis

Kenneth V. Honn L'2'3'4, Dean G. Tang I, Xiang Gao ~, Igor A. Butovich 1'5, Bin Liu 1'¢', Jozsef Timar 1'7 and Wolfgang Hagmann ~'s Departments o F Radiation Oncology, 2 Chemistry, and 3 Pathology, Wayne State University, Detroit, MI48202; 4 Gershenson Radiation Oncology Center, Harper Hospital, Detroit, MI 48201; 5 Laboratory' for Chemical Enzymology, Institute of Bioorganic Chemistry, 1 Murmanskaya St., Kiev, 253660, Ukraine," 7 First Institute of Pathology and Experimental Cancer Research, Semmelweis Medical University, Budapest, Hungary; '~ Deutsches Krebsforschungszentrum, Abt. Tumorbiochemie 0245, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany: ~ Present address: Department o f Medicine, Division of Hematology/Oncology, Duke University, Durham, NC 27710

Key words: eicosanoids, 12-1ipoxygenase, metastasis, adhesion, 12(S)-HETE, protein kinase C

Abstract

Arachidonic acid metabolites have been implicated in multiple steps of carcinogenesis. Their role in tumor cell metastasis, the ultimate challenge for the treatment of cancer patients, are however not well-documented. Arachidonic acid is primarily metabolized through three pathways, i.e., cyclooxygenase, lipoxygenase, and P450-dependent monooxygenase. In this review we focus our attention on one specific lipoxygenase, i.e., 12-1ipoxygenase, and its potential role in modulating the metastatic process. In mammalian cells there exist three types of 12-1ipoxygenases which differ in tissue distribution, preferential substrates, and profile of their metabolites. Most of these 12-1ipoxygenases have been cloned and sequenced, and the molecular and bio- chemical determinants responsible for catalysis of specific substrates characterized. Solid tumor cells express 12-1ipoxygenase mRNA, possess 12-1ipoxygenase protein, and biosynthesize 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid], as revealed by numerous experimental approaches. The ability of tumor cells to generate 12(S)-HETE is positively correlated to their metastatic potential. A large collection of experimental data suggest that 12(S)-HETE is a crucial intracellular signaling molecule that activates protein kinase C and mediates the biological functions of many growth factors and cytokines such as bFGF, PDGF, EGE and AME 12(S)-HETE plays a pivotal role in multiple steps of the metastatic 'cascade' encompassing tumor cell-vasculature interactions, tumor cell motility, proteolysis, invasion, and angiogenesis. The fact that 12-1ipoxygenase is expressed in a wide diversity of tumor cell lines and 12(S)-HETE is a key modulatory molecule in metastasis provides the rationale for targeting these molecules in anti-cancer and anti-metastasis therapeutic protocols.

Introduction

Metastasis is the hallmark of malignancy and the major obstacle to the cure of cancer patients. The overwhelming complexity of this pathological process is evidenced by our as-yet overall paucity of the understanding of the disease despite decades of research by cancer biologists and clinical oncologists.

However, there is no doubt that significant progress has been made in deciphering many of the important molecular events associated with tumor progression and dissemination. Cancer starts with cellular transformation in which germline gene (e.g., oncogenes and tumor suppressor genes) mutations with or without epigenetic deregulation render a normal cell tumorigenic. Transformed cells are

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characterized by uncontrolled cell proliferation in- compatible with normal organ functions. Cell transformation itself, however, will not result in the generation of metastatic tumor cell variants. Further genetic alterations and environmental insults are needed to trigger the dominant tumor cell clones that are able to seed themselves outside the primary lesion. For metastasizing tumor cells, completion of the 'metastatic cascade' is a tremendously challeng- ing endeavor, in which they must survive multiple 'checkpoints' such as vigilant host immune surveil- lance and harsh hemodynamic shear forces and overcome unfavorable biological barriers such as basement membrane. A small population of spreading tumor cells will eventually succeed colonizing a distant organ and forming metastases. Im- portant biological parameters in the metastatic process include angiogenesis, intravasation/extravasation, proteolysis, locomotion/invasion, and cell proliferation [14]. These elements are not isolated but interdependent entities and are regulated by external driving forces exemplified by various growth factors, cytokines, chemokines, motility factors and a plethora of bioactive lipid molecules, among which are arachidonic acid (AA) metabolites.

AA is liberated from membrane phospholipids either by the action of phospholipase A 2 or through the concerted actions of phospholipase C and diacylglycerol lipase or of phospholipase D and phospholipase A 2 [6]. Once released, AA is metabolized through three major pathways. The cyclooxygenase pathway generates prostacyclin (PGI2), thromboxane (TXA2), and various prostaglandins; the lipoxygenase pathway gives rise to a variety of hydroxyeicosatetraenoic acids (HETEs), leukotrienes, and lipoxins; and the cytochrome P-450 monooxygenase pathway form various epoxyeicosatrienoic acids (EET) [for reviews see ref. 6-9]. Arachidonic acid and many of its metabolites have been implicated in multiple steps of tumorigenesis and metastasis [9- 13; also refer to relevant chapters in this issue]. The important role of eicosanoids in modulating cancer metastasis is best illustrated by prostacyclin/thromboxane which in opposite directions regulate tumor cell-vessel wall interactions as well as metastasis [14-17; also see Chapter 11]. More recently, several lipoxygenase products (such as 12- and 15-HETE)

also have been demonstrated to be closely involved in regulating the metastatic process [18-21]. Since numerous comprehensive review articles exist in the literature documenting the general relationship between AA metabolites and tumor cell metastasis, we choose to focus our attention on only one branch of the lipoxygenase pathway, i.e., 12-1ipoxygenase pathway which generates as its major product 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid]. Our elaboration will start with the 12-1ipoxygenase gene structure and then move to the enzymology of the protein. Subsequently, the role of 12(S)-HETE in mediating tumor cell signal transduction and modulating various metastatic phenotypes is discussed. Finally, the potential of using 12-1ipoxyge- nase as a prognostic marker for certain types of human cancer and 12-1ipoxygenase inhibitors as anti- metastasis drugs is discussed.

12-Lipoxygenase: gene structure and regulation

Lipoxygenases insert molecular oxygen into cis, cis- pentadiene-containing lipid molecules generating various hydroperoxides. So far three animal lipoxygenases have been identified; they are 5, 12-, and 15-1ipoxygenases named after the positions where oxygen is inserted to the AA moiety. 5-1ipoxyge- nase metabolizes AA to hydroperoxyeicosatetrae- noic acid (5-HPETE) which is further converted to 5-HETE and leukotrienes. 12-1ipoxygenase (EC 1.13.11.31) metabolizes AA to 12-HPETE which is subsequently transformed to 12-HETE and hepoxi- lins. Similarly, 15-1ipoxygenase catalyzes the reaction that generates 15-HPETE and subsequently 15- HETE and lipoxins.

Unlike 5- and 15-1ipoxygenases, 12-1ipoxygenase exists as isoforms within the same species [22]. Bio- chemical and immunological evidence indicates that 12-1ipoxygenases expressed in bovine leukocytes, platelets, and tracheal epithelium are dis- tinctly different enzymes [23, 24]. Information from cloned 12-1ipoxygenase cDNAs confirmed the existence of different isoforms, cDNAs of 5-, 12- and 15-1ipoxygenase have been cloned from several different species [25-33]. Introduction of human, porcine, bovine or rat 12-1ipoxygenase cDNAs into eu-

karyotic cells or bacteria enables the transfectants to specifically metabolize AA to 12(S)-HETE [26, 31-33], although a small amount [less than 1/10 of the amount of 12(S)-HETE) of 15(S)-HETE also was generated in the case of bovine airway epithelial and rat brain 12-1ipoxygenases [26, 32]. Similar- ly, when cloned human reticulocyte 15-1ipoxyge- nase cDNA was transfected into bacteria, the cell lysate was able to convert AA predominantly to 15(S)-HETE and to a much lesser extent, to 12(S)- HETE [less than 1/10 of the amount of 15(S)- HETE)] [34].

Three types of 12-lipoxygenases have been reported. The first is human platelet-type 12-1ipoxy- genase expressed normally in platelets, HEL (human erythroleukemia) cells, and umbilical vein endothelial cells [35]. Platelet-type 12-1ipoxygenase metabolizes only AA (but not C-18 fatty acids such as linoleic acid) to form exclusively 12(S)-HETE [36]. The second is porcine leukocyte-type 12-1ipox- ygenase which metabolizes both arachidonic acid and linoleic acid thus generating 12(S)-HETE as well as small amounts of 15(S)-HETE [37]. The third type of 12-1ipoxygenase (sometimes termed epithelial 12-1ipoxygenase) has been isolated from bovine tracheal epithelial cells [26] and rat brain [32], which shares more homology with 15-1ipoxy- genase and leukocyte-type 12-1ipoxygenase than with platelet-type 12-1ipoxygenase. This type of 12- lipoxygenase, like reticulocyte 15-1ipoxygenase and leukocyte-type 12-1ipoxygenase, catalyzes the formation of both 12(S)-HETE and 15(S)-HETE [26, 32, 38, 39]. Recently, mouse homologues of both platelet-type and leukocyte-type 12-1ipoxygenases have been reported [40].

When the three types of 12-1ipoxygenases are compared, 323 of the 661 amino acids are found to be identical (Fig. 1). Of these 323 amino acids, 47 residues differ from their corresponding sequences in 15-1ipoxygenase. In this set of 47 amino acids differences between 12- and 15-1ipoxygenases, 4 residues are conserved between the two known 15-1ipox- ygenase sequences [27, 41]. When these 4 amino acids in human 15-1ipoxygenase are mutated to amino acids found in 12-1ipoxygenase, the enzyme catalyzes 12- and 15-oxygenation equipotently [42]. Fur- ther analysis of enzymes with single, double, triple

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and quadruple mutations showed that Met418 is the primary determinant of positional specificity. When Met418 of 15-1ipoxygenase is replaced with a Val (the corresponding amino acid in 12-1ipoxygenase), the resulting enzyme (15-1ipoxygenase, M418V) performs 12- and 15-oxygenation equally thus generating equal amounts of 12- and 15(S)-HETE [42]. No other mutation is found to be responsible for the positional specificity change. The region immediately surrounding amino acid 418 is highly conserved in all mammalian lipoxygenases. Further mutagenesis studies demonstrated that the atoms of the side chain at position 418 are in contact with the substrate [42]. The replacement of the Met with a Val results in a 17% reduction of sidechain vol- ume. This would lead to a shift in substrate binding if the atoms of this side chain were to interact with the substrate. As Met and Val are both hydrophobic amino acids, they could participate in binding a fatty acid substrate. The site responsible for hydrogen abstraction of the wild-type enzyme is close to C-13 on AA, leading predominantly to 15-oxygenation. Presumably, the 15-1ipoxygenase M418V has a deeper substrate-binding pocket than the wild-type enzyme, so the C13 and C-10 of bound AA are equi- distant from the reaction center, thus the alignment between the methylenes and the hydrogen acceptor is imperfect, leading to equal 12- and 15-oxygenation. Thus M418 and its surrounding amino acids are responsible for the positional specificity of lipoxygenases.

When the cDNA and deduced amino acid sequences of 5-, 12-, 15- and soybean lipoxygenases from different sources were compared, it was found that all of lipoxygenases contain a highly conserved sequence in the form of His-(X)4-His-(X)4-His-(X ) 17-His-(X)s-His which represents a potential binding site for non-heme iron. This sequence is found at positions 363-400 for human 5-1ipoxygenase [29, 43], 355-392 for human 15-1ipoxygenase [27, 411 and human platelet 12-1ipoxygenase [31], 357-394 for porcine leukocyte 12-1ipoxygenase [25], and 356- 393 for bovine tracheal epithelium and rat brain 12- lipoxygenases [26, 32; Fig. 1]. Site-directed mutagenesis studies of these conserved histidine residues in human 5-1ipoxygenase by two independent groups have shown that two of five histidine resid-

368

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Fig. 1. Deduced amino acid sequence alignment of 12-1ipoxygenases. Sequences which are identical to that of human platelet-type 12- lipoxygenase are highlighted. Conserved sequences among 6 currently characterized 12-1ipoxygenases are underlined. Conserved histidine residues are marked with an asterisk (*). Conserved isoleucine residues are boxed. Spaces are introduced to allow optimal alignment. Hum: human, pl: platelet-type, Boy: bovine, ep: epithelial, Por: porcine, lc: leukocyte-type, br: brain, Mou: mouse.

ues are impor t an t for the catalytic activity of l ipoxy-

genases [44, 45]. A l though six 12-1ipoxygenase c D N A s have been

c loned f rom five species, its genomic s t ruc ture has only b e e n charac te r ized f rom human , pig, and mouse [35, 40, 46, 47]. All 12-1ipoxygenase genes isolated have 14 exons and the size of the exons, but not introns, are highly conserved a m o n g dif ferent

species. T h e human, porcine, and mouse p r o m o t e r sequences of 12-1ipoxygenase genes have been identif ied by different groups [35, 40, 46, 47], yet little is k n o w n abou t their regulat ion. Mult iple G C boxes were found in the 5' region, raising the possibility of 12-1ipoxygenase as a housekeep ing gene. Howeve r , this is not very likely since 12-1ipoxyge-

nase activity is inducible [33, 48-51].

12-Lipoxygenase Promoter Structures

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Mouse platelet -----IAAGG repeat I [ ~ [ ] ~ lnt

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Fig. 2. Comparison of various 12-1ipoxygenase promoters from different species. The potential regulatory elements and repeat sequences are boxed. Mou: mouse; Por: porcine; Hum: human: Lc: leukocyte; Pl: ptatelet; Int (?): (potential) initiator site.

All 12-lipoxygenase promoters, except the mouse platelet-type 12-1ipoxygenase promoter, contains no typical TATA box. The human platelet-type 12- lipoxygenase promoter contains 4 putative GC boxes, 2 CACCC boxes, 3 AP-2 sites, and 1 glucocorti- cold-responsive element (GRE) [35, 46]. The porcine 12-1ipoxygenase promoter possesses 9 GC boxes and 2 AP-2 sites [47]. In mouse, platelet-type 12- lipoxygenase promoter has 3 GC boxes, 1 TATA site, 1 TATA-like site, and 1 AP-2 site, whereas leukocyte-type 12-1ipoxygenase promoter contains 1 GC box, 1 TATA-like site, and 1 AP-1 site [40]. We have isolated the 12-1ipoxygenase promoter from a human colon carcinoma cell line (Clone A) and the sequence is nearly identical to the published human platelet-type 12-1ipoxygenase promoter sequence with a point mutation affecting one of the four GC boxes (X. Gao and K.V. Honn, unpublished data). Potential regulatory elements in various 12-1ipoxy- genase promoters are compared in Fig. 2. The GRE is activated by glucocorticoid, androgen, mineralo- corticoid and progesterone [52]. The AP-2 site is inducible by TPA, cAMP and retinoic acid [52]. The AP-1 site could be induced by TPA and other protein kinase C (PKC) activators. Indeed, it has been shown that treatment of HEL cells with TPA increases 12-1ipoxygenase mRNA [50] and activity [33]. Furthermore, EGF, glucose, and angiotensin II have been demonstrated to increase 12-1ipoxyge- nase mRNA expression, stimulate 12-1ipoxygenase activity and therefore the production of HETE and HODE (i.e., hydroxyoctadecadienoic acids) [4'8, 49, 51]. Recently, we and others identified authentic

platelet-type 12-1ipoxygenase mRNA in Clone A (human colon adenocarcinoma) cells [53] and keratinocytes [55] and observed that 12-1ipoxygenase mRNA and protein were upregulated by 12-(S)- HETE treatment in clone A cells (Gao and Honn, unpublished data).

12-Lipoxygenase: chemical basis for its enzymatic activity

Lipoxygenases are iron-containing, non-berne enzymes primarily catalyzing the aerobic oxidation of polyunsaturated fatty acids (PUFAs) containing a 1,4-cis, cis-pentadiene fragment yielding the corresponding hydroperoxides as the primary reaction products. It is of great interest to explore the kinet- ics and mechanism(s) of lipoxygenation reactions. The most widespread scheme of lipoxygenase classification is based on the positional specificity of the enzyme toward AA. Depending on the position of the carbon atom being oxidized four major classes of lipoxygenases are discerned, nameley 5-, 8-, 12-, and 15-1ipoxygenases. The experiments carried out with a set of positional isomers of arachidonic acid [38] and linoleic acid [57] suggest that the substan- tial differences in the reaction rate and the set of products formed by different enzymes can be ex- plained by non-optimal alignment of the hydrogen acceptor of the enzyme reaction center and the double allylic methylene group of the substrate. In this connection, it will be significant to know which part of the PUFA molecule is responsible for its posi-

370

tioning in the enzyme's active center, i.e., the methyl moiety or the carboxylic group? The data published by Gardner et al. [58] indicate that it is the carboxylic moiety that plays a crucial role in the substrate orientation. Kuhn et al. [59] also proposed that the wheat lipoxygenase utilized the carboxylic group of the substrate for it to be oriented and aligned in a proper manner. There is, however, recent evidence that the m-end PUFAs might also play an important role in the substrate binding to the enzyme active center [42]. Therefore it seems likely that both parts of a PUFA molecule are essential for the substrate positioning and binding to the enzyme.

At least one Met residue that has been thought to be important in lipoxygenase catalysis is located in a near proximity to the substrate binding center [42, 6062]. It has been shown that the well-known self- inactivation of reticulocyte lipoxygenase is accompanied by oxidation of a single Met to its sulfoxide [60]. Soybean lipoxygenase, when covalently modified with iodoacetic acid or ~-bromstearic acid, but not with iodacetamide (additional evidence of the important role of carboxylic group in the substrate binding) lost its activity in a time- and dose-dependent manner, suggesting that the protonated carboxyl group of iodacetic acid is essential for the inhibitor positioning [61]. Taken together, these results indicate that the Met residue under consideration is located near the carboxyl-binding site of lipoxygenase and its catalytic center. The crucial role of the Met418 in positioning of PUFA in mammalian 12- and 15-1ipoxygenases was also postulated by Sloane et al. [42], as discussed in the preceding section.

All lipoxygenases so far reported are non-heme iron-containing enzymes. Four to five conserved histidines found in mammalian as well as plant lipoxygenases are thought to serve as ligands for the iron ion [63-66]. Usually Fe/apoenzyme ratio approaches 1, depending on the source of the enzyme and methods used to extract and purify it. It is known that the iron in the enzyme's active center can be in high-spin ferric (Fe 3+) or ferrous (Fe 2+) forms. The redox state of the Fe 2+ can be easily changed by addition of micromolar range concentrations of hydroperoxides of fatty acids [67], hydrogen peroxide [68] or even, as proposed recently,

by oxygen [69]. Ferric iron undergoes reduction under the influence of some reductants, and specifically by PUFAs themselves [70]. The most probable state of iron in plant cells is the ferrous form, imply- ing that the enzyme must be activated by an appropriate oxidant to be able to oxidize PUFAs. In contrast, porcine leukocyte 5- and 12-1ipoxygenases, at least partly, are extracted from the cells and further purified in the ferric form, as revealed by direct EPR experiments [71, 66]. Thus, it appears that they do not need a PUFA hydroperoxide to be converted into the catalytically active form.

Considering the mechanism of aerobic lipoxygenase reaction and not going beyond the universal 1,4-cis, cis-pentadiene moiety one has to take into account the following scheme which has been proposed to describe the course of events in the enzyme catalytic center:

LO-Fe(III) LO-Fe(II)

enzymatic, aerobic . .

R- (w) (m)

HOO anaerobic

aerobic ' Mixture of (R,S)-isomers R-R

(V)

The PUFA (I), which is stereospecifically deprot- onated by some enzyme-bound nucleophile B (-) to form an enzyme-bound carbon-centered free radical (II), undergoes either i) stereoselective oxygenation giving the corresponding hydroperoxide (III), or, under anaerobic conditions, ii) subsequent dissociation (IV) and recombination resulting in the formation of dimers of PUFA (V). Sometimes, a side reaction leading to the formation of the race- mic mixture of (R,S)-hydroperoxides (VI) occurs as the result of dissociation of complex (II) in the presence of molecular oxygen and further non-enzymatic oxygenation of radical (IV).

Most lipoxygenases are capable of further trans-

formations of hydroperoxides. It has been shown [70, 72] that mammalian as well as plant lipoxygenases catalyze transformation of hydroperoxide 0II) to ketones (VII) (so-called 'anaerobic' reaction):

LO ) >o H20

III VII

and double oxygenation of tri- or tetraenoic acids [73]. It recently has been demonstrated that in the presence of certain proton donors lipoxygenases can possess hydroperoxidase activity [74], and more intriguingly, can oxidize a very unusual substrate, 12-keto-(9Z)-octadecaenoic acid methyl ester (VI- II) yielding the methyl ester of 9,12-diketo-(10E)- octadecaenoic acid (IX) [75]:

O

02

VIII IX

The enzyme activity toward 9(E), 12(Z)-octade- cadienoic acid, a rotational isomer of linoleic acid, is a well-known phenomenon reported by Funk et al.

[76]. Therefore, lipoxygenases may not require, for their enzymatic catalysis, the presence of double allylic methylenes in their substrates. It appears that most types of lipoxygenases can also catalyze the formation of epoxide (X) from the corresponding hydroperoxide (III) [63-65]:

OOH

II!

Lo ~

7 H20 x

In addition, 12- and 15-1ipoxygenases can also cleave their primary reaction products to give an alkane (XI) and an aldehyde (XII) [77]:

371

LO H ~ > + Alkane

H20 O XII Xl

III

Finally, peroxidase-like activity of lipoxygenases has also been demonstrated, but appropriate proton donors are required [74]. The mechanism of the enzymatic oxidation of esterified (e.g., incorporated into phospholipids) PUFAs is intrinsically the same as has been described for free PUFAs.

12-Lipoxygenase: modulation of expression, activity, and intracellular distribution by extra- and intracellular factors

The intracellular 12-1ipoxygenase is subject to modulation and regulation at various steps encompassing gene transcription, presence of active enzyme, and its inactivation. The susceptibility of 12-1ipoxy- genase to these modulatory actions varies between the enzymes from different cell types. Thus the contribution of any or all of such regulatory factors to the overall activity of 12-1ipoxygenase in a specific cell type deserves detailed examination.

Expression of the mRNA for 12-1ipoxygenase has been reported for human platelets [31, 33], leukocytes [33], endothelial cells [35], epidermal cells [78], and also for various human tumor cells including erythroleukemia (HEL) cells [33, 35], colon carcinoma (Clone A) cells [53], epidermoid carcinoma A431 cells [48], rat Walker carcinosarcoma (W256), murine Lewis Lung carcinoma (3LL) and amelanotic melanoma (B16a) cells [53]. The presence of 12-1ipoxygenase mRNA, however, is not paralleled in all cases by the existence of a detectable amount of 12-1ipoxygenase protein or activity. In Clone A and cultured 3LL cells, 12-1ipoxygenase mRNA is detectable after amplification by RT-PCR [53], but the 12-1ipoxygenase activity or protein could not be demonstrated with certainty (Hagmann and Honn, unpublished observations). Thus, at least in some cell types one possibility for the regulation of 12-1i- poxygenase appears to exist at the post-transcrip- tional level. On the other hand, the expression of

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12-1ipoxygenase mRNA in a specific cell type can be modulated. Up-regulation of 12-1ipoxygenase mRNA within 10-18 h is elicited in A431 cells by EGF [48]. In aortic smooth muscle cells, glucose and angiotensin II also cause a 2-3 fold increase in 12-1ipoxygenase mRNA expression [51]. In HEL cells, conflicting evidence was reported showing both up-regulation [50] and down-regulation [79] of 12-1ipoxygenase mRNA after treatment of the cells with phorbol ester TPA. The reason for this discrepancy is yet unclear, but nevertheless stresses the notion that the 12-1ipoxygenase mRNA expression in these cells is subject to regulation by extracellular factors. Interestingly, an additional way to induce 12-1ipoxygenase mRNA expression apparently exists in Clone A cells, where 12-HETE, generated by 12-1ipoxygenase activity and subsequent reduction, can enhance 12-1ipoxygenase mRNA expression within a few hours (Gao and Honn, unpublished observations), thus arguing for the existence of a possible positive feedback regulation of 12-1ipoxy- genase expression by the product of its own enzymatic activity.

In cells containing enzymatically active 12-1ipox- ygenase, the intracellular distribution of this activity varies between different cells and does not nec- essarily correspond with the respective distribution of the 12-1ipoxygenase protein. The subcellular distribution of 12-1ipoxygenase activity ranges from a predominantly cytosolic compartmentation in platelets, leukocytes, and epithelial cells [24, 80, 81] to an exclusive or preferential membrane-associated activity in A431, HEL, freshly isolated 3LL, and epithelial cells [49, 79, 82-85]. In accordance with their inducing effect on 12-1ipoxygenase mRNA expression, extracellular factors such as glucose, angiotensin II, and EGF also cause an increase in 12-1ipoxy- genase activity in the respective cells or subcellular fractions [49, 51]. As to microsomal membranes from HEL cells, conflicting data document either an increase or a decrease of 12-1ipoxygenase activity following TPA treatment of cells [33, 79, 84]. On the other hand, deprivation of EGF-responsive A431 cells of this growth factor rapidly results in the loss of 12-1ipoxygenase activity, whereas re-addition of EGF or serum to such starved cells immediately re- stores the 12-1ipoxygenase activity in some cells

[189]. Interestingly, 12-1ipoxygenase was reported to be differentially sensitive to activating or inhibitory factors depending on its intracellular localization. Glutathione (GSH) concentrations above 1.5 mM inhibit cytosolic 12-1ipoxygenase activity in platelets, whereas the membrane-associated 12-1i- poxygenase activity in HEL or ovine epithelial cells is already inhibited by 1 mM or 0.1 mM GSH, respectively [83, 84]. Analogously, dithiothreitol (DTT) inhibits the membrane-associated 12-1ipox- ygenase activity from HEL cells [196]. Inversely, hydroperoxides such as 13-HPODE do not influence membrane-associated 12-1ipoxygenase activity [79, 83], but have been demonstrated to be sufficient for activating cytosolic 12-1ipoxygenase activity which is inactive in the absence of 13-HPODE [84].

The subcellular distribution of the 12-1ipoxyge- nase protein in some tumor cells does not parallel the intracellular localization of their 12-1ipoxyge- nase activity. In 3LL cells, the 12-1ipoxygenase protein is localized predominantly in the cytosol [53, 85], as is their 12-1ipoxygenase activity [82, 85]. In A431, HEL, W256, and B16a cells, however, the predominantly cytosolic 12-1ipoxygenase protein is enzymatically far less active than the minor amount of membrane-associated 12-1ipoxygenase protein [53, 85] suggesting a tight regulation of 12-1ipoxyge- nase activity in the cytosol of these cells. In addition, the subcellular distribution of 12-1ipoxygenase activity and protein can change upon stimulation of cells, suggesting a translocation of the enzyme protein as a physiologic event during its activation process in cells [84-86]. Translocation of an enzyme from cytosol to membranes during its activation is known to occur with many enzymes such as PKC [87], PLC, PLA 2 [88, 92], diacyl- and 2-monoacyl- glycerol lipases [89], and 5-1ipoxygenase [90], whereas the reported 8-1ipoxygenase in murine skin apparently does not translocate to membranes in a Ca2+-dependent manner [91]. In platelets, such a Ca2+-induced stimulation was shown to result in an increase in membrane-associated 12-1ipoxygenase activity and a concurrent loss of its activity from the cytosol [86]. This Ca 2+-, thrombin-inducible shift in the subcellular localization of 12-1ipoxygenase activity [86] is paralleled by a concommitant translocation of the 12-1ipoxygenase protein (Hagmann

and Honn, unpublished observations), and the membrane-associated 12-1ipoxygenase can be reco- vered as fully active enzyme by decreasing the free Ca 2÷ concentration [86]. In contrast to platelets, a Ca2+-induced loss of cytosolic 12-1ipoxygenase activity in 3LL cells is reflected by translocation of 12- lipoxygenase protein, but this process results in a membrane-associated enzyme which is not fully active or becomes inactivated shortly after translocation to the membrane site [85]. In HEL cells, Ca 2+ also induces a concentration-dependent decrease in the residual cytosolic 12-1ipoxygenase activity and a concomitant translocation of activity to membranes [84].

The discrepancy in some cells between distribution of 12-1ipoxygenase protein and its activity in subcellular compartments suggests that the enzyme is inhibited in the cytosolic compartment. Such an inhibitory effect by cytosolic constituents on 12-1i- poxygenase activity has been suggested previously [49, 83]. These earlier studies, however, reported a cytosol-dependent decrease in specific activity rather than in total or relative 12-1ipoxygenase activity disregarding the fact that the addition of cytosol to the membrane fraction affects markedly the specific activity by increasing the protein content of the sample tested. However, in HEL cells addition of cytosol to the enzymatically active membrane fraction does not alter its relative 12-1ipoxygenase activity, whereas its specific 12-1ipoxygenase activity decreases dose-dependently with added cytosol [84].

12(S)-HETE: involvement in multiple steps of cancer metastasis

12(S)-HETE is the major AA metabolite of 12-1i- poxygenase. Many different types of normal cells including platelets, neutrophils, macrophages, endothelial cells, and smooth muscle cells can synthesize 12(S)-HETE [7, 51, 158]. The physiological functions of this eicosanoid are not fully characterized, but accumulated data indicate that it is involved in a wide-spectrum of biological activities such as stimulating insulin secretion by pancreatic tissue, suppressing renin production, chemoattract-

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ing leukocytes, and facilitating the attachment of macrophages to rat glomeruli during inflammation (reviewed in Ref. 7). 12(S)-HETE also has been observed to reduce prostacyclin biosynthesis by vascular endothelial cells [93] and play a vital role in platelet activation and aggregation [94]. More recently, 12(S)-HETE is found to be the most prominent AA metabolite in menstrual blood [95] and in intrauterine tissues [96], however, the biological signifi- cance of these findings is not yet clear.

As discussed in the previous section, a variety of tumor cells including solid tumor cells express 12- lipoxygenase mRNA and protein [48, 49, 53, 84, 85, 87, 97]. RT-PCR together with immunoblotting revealed that cultured solid tumor cells from human, rat, and mouse express platelet-type 12-1ipoxyge- nase mRNA and protein [53, 84, 85, 87]. Reverse phase HPLC identified 12(S)-HETE as the major metabolite of AA in these tumor cells which is confirmed by chiral phase HPLC and GC/MS analysis [97, 98]. Several lines of evidence suggest that the ability of tumor cells to synthesize 12(S)-HETE is a strong correlate of their metastatic potential. First, subpopulations of B16 amelanotic melanoma (B16a) have been isolated by centrifugal elutriation [99] that demonstrate differential metastatic capa- bilities. The low metastatic B16a (i.e., LM180) cells biosynthesized similar amounts of 12(S)-HETE and 5-HETE, while high metastatic B16a cells (i.e., HM340) generated predominantly 12(S)-HETE with only small quantities of 15-, 11-, and 5-HETEs [97]. Furthermore, HM340 cells synthesized 4 times more 12(S)-HETE than LM180 cells when equal amounts of substrates were supplied [97]. The generation of higher amount of 12(S)-HETE in HM340 cells appears to result from the presence of a higher level of 12-1ipoxygenase mRNA in these cells [195]. Second, the correlation of 12(S)-HETE production and metastatic potential also was evaluated in several other tumor cell systems, i.e., Dunning rat prostate carcinoma (AT2.1 and GP 9F3, low metastatic cell lines; MAT Lu and MLL, high metastatic lines), murine B16 melanoma (F1, low metastatic; F10, high metastatic), and murine K-1735 melanoma (C1-11, low metastatic clone; M1, high metastatic clone). In all these experiments it was observed that the high metastatic tumor cell lines generate signif-

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icantly higher level of 12(S)-HETE than low metastatic counterparts [195]. Third, tumor cell adhesion to fibronectin provokes a spike of 12(S)-HETE generation within 10 rain indicating a late-type signaling activation of tumor cell 12-1ipoxygenase [112]. Morphological studies demonstrated that tumor cell spreading on fibronectin is followed by 12-1i- poxygenase translocation to the cell surface [112]. Fourth, adhesion of tumor cells to vascular endothelium is accompanied or immediately followed by a surge of 12(S)-HETE biosynthesis by tumor cells, which was correlated with tumor cell-induced endothelial cell retraction [98, 100]. LM180 B16a cells generated little 12(S)-HETE upon adhesion and did not induce endothelial cell retraction. In contrast, HM340 B16a cells adhering to endothelium biosynthesized large amounts of 12(S)-HETE and induced prominent retraction of endothelial cell monolayers [98]. Fifth, pretreatment of tumor cells with a select platelet-type 12-1ipoxygenase inhibitor, BHPP (N-benzyl-N-hydroxy-5-phenylpenta- namide; 53) ~ . . . . d0se-dependently inhibited adhesion- induced tumor cell biosynthesis of 12(S)-HETE and endothelial cell retraction [98], platelet-enhanced tumor cell-induced endothelial cell retraction [100], tumor cell (i.e., HM340 B16a cells) adhesion to endothelium and matrix [53, 97], and lung colonization by HM340 B16a cells [97]. Taken together, these data implicate 12-1ipoxygenase and 12(S)- HETE production as important determining parameters of tumor cell metastasis. In the subsequent discussions, we will analyze in detail the versatile modulatory role of 12(S)-HETE in various intermediate steps of metastasis.

A. Effect of l2(S)-HETE on tumor cell interactions with extracellular matrix (ECM)

Tumor cell - ECM interactions are mediated by adhesion receptors such as integrins, non-integrins and proteoglycans where integrins are considered to be the predominant matrix receptors [18, 101, 102]. Tumor cell - ECM interactions are key and multiple events during tumor progression; i.e. in the release of tumor cells from the primary site, in the locomotion and invasion of dissociated tumor cells

in interstitium, in intravasation and extravasation, and in the eventual establishment of the metastatic nodule. It is conceivable that the regulation of the surface expression of these matrix receptors has a great impact on tumor cell adhesive potentials.

Integrin receptors, after synthesis, are stored in tumor cells as well as in normal cells intracellularly in so called adhesosomes [103] and tumor cells have a large pool of intracellular receptors [104]. How- ever, in normal cells, freshly synthesized and glycosylated integrins are not competent for their full range of ligand binding and signaling functions. Ad- hesion process initiated by either soluble or solid surface-bound ligands triggers cell activation. Li- gand binding to the surface exposed integrins induces conformational change of the receptors increasing the binding potential and modulating their binding specificity. In parallel, such physical alterations in the receptors induce signal transmission to the cell interior by activation of a receptor associated tyrosine kinase, FAK (focal adhesion kinase; 105-107). As a consequence, cytoskeletal proteins become associated with the cytoplasmic domains of the integrins and this process immobilizes and con- centrates the receptors to the areas of cell - matrix contacts called focal adhesion plaques. During the cytoskeleton-dependent process the cell shape changes dramatically from round to spread.

Several extracellular factors, examplified by ECM-derived soluble ligands and ECM-bound cytokines (characteristically these are heparin-binding cytokines such as TGF-[~, FGFs, VEGE etc.) may regulate or alter the adhesive properties of tumor cells. In the case of tumor cell - vessel wall interactions other cellular participants such as platelets and endothelial cells modulate the adhesion process by releasing cytokines and metabolites of AA to the micromilieu of the tumor cells. There- fore, a possibility is raised that the metabolites of AA generated in the extracellular space of tumor cells may modulate the tumor adhesiveness and thus tumor cell adhesion to ECM. Systematic studies indicated that 12(S)-HETE is able to stimulate metastatic murine tumor cell adhesion to fibronectin and subendothelial matrix in a dose and time- dependent fashion, with a maximal effect observed at 0.1 ~M of 12(S)-HETE 15 min after stimulation

[104, 108]. Analysis of the mechanism of the 12(S)- HETE enhanced tumor cell adhesion indicated an increased surface expression of integrin receptors cdIb133 after 12-(S)-HETE treatment [104, 108]. In the murine tumor cell lines studied (3LL and B16a) the increased adhesiveness was due to overexpres- sion of cdIb133 cytoadhesin [109,110], a promiscuous matrix receptor physiologically expressed by platelets and cells of the hematopoietic lineage. Interest- ingly, TPA, a direct activator of PKC, mimicked the effect of 12(S)-HETE [108] suggesting a PKC-mediated pathway for the exogenous 12(S)-HETE effect (see below).

The 12(S)-HETE effect on tumor cell integrin expression was not due to an increased transcription of the gene or increased protein translation. Rather it was due to a rapid receptor translocation to the cell surface from the intracellular pool [104, 111]. These studies further indicated that the 12(S)- HETE effect on integrin upregulation and enhanced adhesion both depend on rnicrofilaments and intermediate filaments [104, 111].

12(S)-HETE does not only stimulate tumor cell adhesion to fibronectin but also promotes tumor cell spreading on this matrix protein, by inducing reestablishment of the filamentous cytoskeleton system and enhancing the formation of focal adhesion plaques [112]. This process appears to be dependent on the function of PKC [112]. The dose and time-frame of the 12(S)-HETE effect on spreading followed those observed previously for adhesion [112].

B. Effect o f exogenous 12(S)-HETE on tumor cell motility

Tumor cell motility is regulated by para- and autocrine cytokines including SF (scatter factor), MF (motility factor) and AMF (autocrine motility factor) [113]. These cytokines act through their corresponding signaling receptors expressed at the cell surface of tumor cells. AMF was isolated as the first autocrine motility cytokine produced by tumor cells [114]. A receptor for AMF, gp78, was later identified and sequenced [115]. These studies revealed a trans-

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membrane protein with considerable homology to p53 [115].

Hydroxy fatty acid metabolites of AA including 12-(S)-HETE have been shown to be involved in chemotactic movement of leukocytes where they serve as chemoattractants [7]. Therefore there exists a possibility that 12-(S)-HETE also may modulate tumor cell movement. Studies on murine melanoma cell lines with different metastatic potential (B16a, K1735-C1.11 and K1735 M1) indicated that 12(S)-HETE induces tumor cell motility comparable to AMF itself [116]. Subsequent mechanistic studies demonstrated that 12-(S)-HETE, similar to its effect on tumor cell integrin receptor expression, increases gp78 expression at the cell surface in a dose and time-dependent manner [116]. The increased gp78 expression is due to translocation of the receptor to the cell surface from an intracellular tubulovesicular (TVS) structure positive for lysoso- real markers [117]. Studies also indicated that this TVS pool is closely associated with microtubules and intermediate filaments, therefore it is reasonable to assume that the increased AMF receptor expression after 12(S)-HETE stimulation is also a cytoskeleton-dependent process.

More recently, 12(S)-HETE also was observed to increase the invasive potential of AT2.1 rat prostate tumor cells [116, 118]. The tumor cell motility (as assessed by phagokinetic track assay) and invasion (as assessed by Matrigel invasion assay; Fig. 3) were significantly augmented by 12(S)-HETE [118]. The 12(S)-HETE effect was dramatically inhibited by a selective PKC inhibitor calphostin C as well as by a cell membrane-permeable Ca2+-chelator BAPTA [118], suggesting the involvement of PKC (see below). Whether the 12(S)-HETE-promoted motility involves gp78 and whether 12(S)-HETE-augmented invasion in the rat tumor cell system involves activation of proteolytic enzymes are at present unclear.

The reciprocal relationship between 12-1ipoxyge- nase/12(S)-HETE and AMF/gp78 appears to be far more complicated than presented above. 12(S)- HETE apparently promotes an AMF-comparable and gp78-mediated tumor cell motility. On the other hand, gp78 also has been observed to stimulate 12(S)-HETE production in highly metastatic

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Fig. 3. 12(S)-HETE enhances the invasive capacity of rat prostate carcinoma cells. The invasion assay was performed using the Biocoat Matrigel invasion chambers (Collaborative Res., Bedford, MA). Fifty thousand of rat AT2.1 prostate adenocarcinoma cells treated with either ethanol (a) or 0.1 pM 12(S)-H ETE (b) were placed into the upper chamber in 200 pl of serum free RPM11640 medium. To the lower chamber either RPMI or 3T3 cell conditioned medium was added. The cell invasion was quantitated as described [118]. It is obvious that 12(S)-HETE treatment (b) resulted in a significant increase in the invasion of AT2.1 cells, x 400.

murine melanoma cells [116]. More interestingly, AMF, which stimulated the motility of the high- (M1) but not low-metastatic (C1.11) variants of the K1735 murine melanoma, increased expression of 12-1ipoxygenase protein in M1 sublines exclusively. Also, only the M1 (but not C1.11) cells responded to AMF stimulation with increased endogenous 12(S)- HETE production and 12(S)-HETE, like AME only increased the motility of M1 cells [190]. Taken together, these data suggest that 12-1ipoxygenase and 12(S)-HETE may be important intermediate signaling elements in AMF-gp78 mediated tumor cell motility.

C. Effect of exogenous 12(S)-HETE on the release of lysosomal enzymes

Earlier studies on various normal cells indicated that 12(S)-HETE induces lysosomal enzyme or hor- mone secretion [7]. Furthermore, lipoxygenase metabolites have been shown to be involved in collagenase IV release from tumor cells (reviewed in ref. 119). Secretion of proteins involves a so-called se- cretory mechanism, where the glycosylated protein is transported to the plasma membrane by the se- cretory pathway involving transport vesicles. Lyso- somal enzymes utilize a mechanism which directs these proteins to lysosomes by recognizing the M6P residues on the protein (M6P-R). Lysosomal enzymes are released from these vesicles after intra- or extracellular signaling. Tumor cells produce a wide range of proteolytic enzymes by which they di- gest the surrounding ECM. These degradative enzymes include collagenase IV, interstitial collage- nases, heparinases, urokinase and cathepsins with variable substrate specificities [120-122]. Secretion of these enzymes from tumor cells provides a means by which tumor cells can cross tissue boundaries. In normal cells, the majority of such proteolytic enzymes are stored intracellularly and released only after appropriate stimulation. However, in tumor cells lysosomal enzymes, especially cathepsins, were found to be associated constitutively with the cell surface [121-123].

It is known that the translocation of lysosomal proteins is mediated by cytoskeletal elements, pri-

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marily by microtubules. The demonstrated involvement of 12(S)-HETE in the regulation of receptor (such as integrin cdIb[33) translocations through cytoskeleton-dependent mechanisms prompted us to study its effect on the surface expression and release of cathepsin B from tumor cells. At a concentration which is optimal for increased adhesiveness and motility of tumor cells (i.e., 0.1 gM), 12(S)- HETE was observed to induce cathepsin B release from highly malignant cells, i.e., B16a murine melanoma cells and MCF10AneoT hmnan mammary carcinoma cells [119, 122, 124]. Exogenous 12(S)- HETE first triggers the release of mature cathepsin B and then later the immature form, suggesting that the mature form of the enzyme is at or near the cell surface while the immature form is stored intracellularly. Subsequent morphologic studies indicated that, following 12(S)-HETE stimulation, the intracellular cathepsin B-containing vesicles are directed to specialized cell surface areas, i.e., lamellipodia and filopodia [119]. Analysis of the release process induced by 12-(S)-HETE indicated that, similarly to its effect on tumor cell adhesion and motility, the stimulated release of cathepsin B is also a PKC-dependent process [119].

D. Effect of exogenous 12(S)-HETE on the tumor cell infrastructure and cytoskeleton

It is clear from the above discussions that 12(S)- HETE has a regulatory effect on complex cellular processes associated with cytoskeletal functions, such as adhesion to and spreading on ECM proteins, motility, lysosomal enzyme release and receptor translocation to the cell surface. Therefore, we have analyzed the effect of exogenous 12(S)-HETE on cytoskeleton in tumor cells [125, 126]. These studies indicated that 12(S)-HETE induces translocation of organelles from the perinuclear space to the cell periphery paralleled by a reversible rearrangement of cytoskeletal filaments [125]. The ear- liest event post 12(S)-HETE treatment is a de- creased labeling of cytoplasmic stress fibers with a concomitantly enhanced cortical actin labeling. These alterations in microfilaments are accompanied or immediately followed by microtubule poly-

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ermization and vimentin intermediate filament bundling and also by rearrangements of the focal adhesion protein vinculin [125, 126]. The 12(S)- HETE effect on tumor cell cytoskeleton is PKC-dependent since pretreatment of tumor cells with a selective PKC inhibitor, i.e., calphostin C abolished the 12(S)-HETE effects [125]. Interestingly, 12(S)- HETE-induced cytoskeletal alterations generally abate by ~ 30 min. However, simultaneous treatment of tumor cells with 12(S)-HETE and okadaic acid, a general serine/threonine protein phosphatase inhibitor, prevented disappearance of the 12(S)-HETE effects by 30 min [126]. These observations thus raise the possibility that a phosphatase (s), activated due to either the direct effect of 12(S)- HETE or homeostatic mechanisms, is responsible for dampening the 12(S)-HETE-triggered, PKC- dependent, and phosphorylation-mediated cytoskeleton rearrangement.

E. Effect of12 (S)-HETE on vascular endothelial cells: induction of endothelial cell retraction and modulation of integrin expression and cell proliferation

Tumor cell - endothelial cell interactions constitute one of the most important factors in the organ preference of metastasis. Specific interactions of metastasizing tumor cells with end organ endothelial cells will, to a large degree, dictate the route of metastasis. In the vasculature, migrating tumor cells, uti- lizing cell surface adhesion molecules, first attach loosely to the intact endothelial cell monolayers by recognizing counterpart adhesion receptors on the latter cell type. This initial cell-cell adhesion leads to, by certain unknown mechanisms, the activation of tumor cells and/or endothelial cells (or other host cells such as platelets). Cellular activations result in an enhanced surface expression of adhesion receptors and/or a functional maturation in the binding competence of these adhesion molecules, leading to a tighter bonding of tumor cells to endothelium. This whole process of tumor cell - endothelium interactions has previously been analyzed at the molecular level with the 'docking and locking' hypothesis [18]. The hypothesis dissects tumor cell in-

teractions with target organ endothelial cells into two basic interdependent but distinct steps, as illustrated in Fig. 4. The early interaction, i.e., the 'docking', occurs between the 'rolling' tumor cells and underlying endothelial cells and generally is mediated by relatively weak and transient adhesion mechanisms involving carbohydrate-carbohydrate (such as Le×-Le x and glycosphingolipid-glycosphingolipid; 191-192), carbohydrate-selectin (such as sLeX-E selectin), and protein-protein (such as PE- CAM-1-PECAM-I; 145) recognition (Fig. 4; see ref. 18 for detailed account). The initial tumor cell-endothelial cell interactions result in the activation of either cell type or other host cells such as platelets, which leads to increased surface expression and/or functional 'maturation' of other adhesion molecules, typically integrins, and finally to the second phase, tighter tumor cell adhesion, i.e., the 'locking' step (Fig. 4). The transition from the 'docking' stage to the 'locking' stage relies on the generation from both tumor cells and host cells (i.e., leukocytes, platelets, endothelial cells, etc) of various bioactive mediators including cytokines, chemokines, autocrine factors, as well as lipid molecules examplified by 12(S)-HETE (Fig. 4; 18 and references therein).

After tumor cells have established tight, physical connections with endothelial monolayers, tumor cells spread and then exit from the vessel by inducing endothelial cell retraction. Although some types of tumor cells may extravasate by intravascu- lar proliferation followed by rupture of the vessel wall, the majority of experimental (morphological as well as functional) studies both in vivo [127] and in vitro [128, 129] have demonstrated that vascular endothelial cells retract prior to the extravasation of most tumor cell types. Therefore, tumor cell induced endothelial cell retraction has been proposed to facilitate tumor cell metastasis. Unfortunately, we are still far from understanding the molecular mechanisms underlying tumor cell-induced endothelial cell retraction. Although many factors including vasoactive substances (histamine, bradyki- nin, serotonin, etc), lipid mediators (LTC4, PAF, etc), thrombin, TNF-c~, oxygen radicals, neutral proteases, endotoxins, and 5'-radiation have been shown to increase endothelial cell permeability and induce physical endothelial cell retraction [130-

"Docking" Transition "Locking" 379

EC

*Weak and transient binding

*Mediated mainly by carbohydrate-carbohydrate recognition

*Activation of tumor cells and host cells (platelets, leukocytes, endothelial cells, etc)

*Generation of inflammatory cytokines, chemokines, and bioactive lipids

*Expression of inducible adhesion molecules such as selectins, ICAM, and VCAM

*Tighter tumor cell adhesion to endothelial cells

*Mediated principally by integrin receptors

*Integrins' are the major signal transducers for the subsequent molecular events (endothelial cell retraction, interaction with ECM, proteolysis, cell locomotion and invasion

Fig. 4. The 'docking and locking' hypothesis. Tumor cell interactions with vascular endothelial cells can be dissected into an initial phase of weak adhesion (i.e., 'docking') and a later phase of tight adhesion (i.e., 'locking'). Differential involvement of specific subsets of adhesion molecules in these two phase have been demonstrated (see text and ref. 18 for details). The transition from the 'docking' phase to the 'locking' stage is mediated via a large array of bioactive mediators including 12(S)-HETE. TC, tumor cell; EC, endothelial cells.

132], the biological mediators responsible for tumor cell induced endothelial cell retraction largely re- main elusive.

Five years ago, our laboratory presented the first experimental evidence that 12(S)-HETE, a biolog- ically relevant molecule derived from AA metabolism, is capable of inducing a non-destructive, reversible retraction of cultured endothelial cell monolayers [133]. 12(S)-HETE induced endothelial cell retraction resulted in an enhanced tumor cell adhesion to exposed subendothelial matrix [133] and could be inhibited or blocked by prostacyclin or by 13(S)-HODE [134], reminescent of the bidirectional control of tumor cell alIb[33 integrin expression by 12(S)-HETE/13(S)-HODE [108] and of tumor cell - platelet interactions by thromboxane A2 and prostacyclin [14-17]. Subsequent studies established that 12(S)-HETE also induces retraction of microvessel endothelial cells [135-137]. More importantly, we recently demonstrated that tumor cell adhesion to microvessel endothelium induced a non-denuding retraction of endothelial cell monolayers [98], which, in many aspects, mimicked the 12(S)-HETE-induced endothelial cell retraction. Tumor cell induced endothelial cell retraction can be potentiated by homologous platelets [100], thus

providing an experimental rationale for the platelet contribution to tumor cell metastasis. Further in- vestigations revealed that both platelet-augmented and tumor cell induced endothelial cell retraction were mediated through 12(S)-HETE [98, 100]. These observations provide strong evidence for the possible scenario that in v ivo 12(S)-HETE is produced at high concentrations in the localized areas of tumor cell-platelet-endothelial cell interactions. This de n o v o synthesized 12(S)-HETE, most prob- ably by tumor cells and more importantly from platelets, may be transferred to endothelial cells through transcellular metabolism to induce endothelial cell retraction, since there has been experimental evidence that small molecules can be transferred from tumor cells to endothelial cells [138].

Endothelial cell retraction can be affected by a variety of mechanisms dependent on different caus- ative agents. Thus, histamine-induced endothelial cell retraction is mediated by ATP-dependent microfilament rearrangement [139]. By contrast, thrombin-induced endothelial cell gap-formation is dependent on the levels of intracellular cAMP [140]. Recently, myosin light chain kinase has been implicated in endothelial cell retraction caused by many substances [141]. Our previous work demon-

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strated that 12(S)-HETE is an activator of PKC in tumor cells [87]. Recently, we have observed that endothelial cell retraction induced by 12(S)-HETE is also a PKC-dependent process [135-137, 142]. 12(S)-HETE activates PKC in endothelial cells, which subsequently phosphorylates several major cytoskeletal elements including myosin light chain, actin and vimentin, leading to a reorganization of the cytoskeleton network (microfilament, intermediate filament, and focal adhesions) and apparent endothelial cell retraction.

12(S)-HETE induced endothelial cell retraction may also involve alterations in adhesion molecules that normally are concentrated at cell-cell junc- tions. Specically, integrin av[33 and PECAM-1 (platelet endothelial cell adhesion molecule-l), an immunoglobulin supergene family member that is expressed on platelets, vascular endothelial cells, certain subtypes of neutrophils, as well as in some solid tumor cells [143-145], are localized to the cell junc- tional areas in confluent endothelial cell cultures. 12(S)-HETE treatment results in a series of well- defined changes in c~v133-containing focal adhesions, leading to an eventual decrease in av[~3 localization to focal adhesion plaques at both cell body and cell-cell borders [135,136]. In contrast to its effect on integrin o~vl33,12(S)-HETE does not appear to alter the normal cellular distribution patterns of another integrin receptor, i.e., c~5131, an authentic fibronectin receptor. The 12(S)-HETE induced reorganization of c~v133-enriched focal adhesions resem- bles its effect on vinculin and again appears to be dependent on PKC activation [135,136]. PECAM-1 has been proposed to be involved in maintaining the integrity of endothelial cell monolayers [143]. Treatment of post-confluent endothelial cell cultures with 12(S)-HETE dispersed cell junction-localized PECAM-1 molecules to the whole cell body (Tang and Honn, unpublished observations). Whether this alteration of PECAM-1 is a trigger or a consequence of 12(S)-HETE induced endothelial cell retraction remains to be established.

Interestingly, 12(S)-HETE also modulates the surface expression of integrin receptors on endothelial cells. 12(S)-HETE stimulation upregulates the surface expression of integrin av[33 (but not o~5131) in both large vessel [146] and microvessel

[136] endothelial cells. The increased czv[33 surface expression appears to be concomitant with a decrease in ~v[33-enriched focal adhesions [136] and is a posttranscriptional, PKC- and cytoskeleton-dependent process [147]. The end result of 12(S)- HETE enhanced surface expression of (zvl]3 integrin receptors is increased tumor cell adhesion to the activated endothelium [136, 146, 147].

12(S)-HETE may as well be involved in regulating endothelial cell growth. Early work demonstrated that the DNA synthesis as well as proliferation of cultured fetal bovine aortic endothelial cells were inhibited by nordihydroguaiaretic acid (NDGA), a general lipoxygenase inhibitor, but not affected by the cyclooxygenase inhibitor indomethacin [149], suggesting lipoxygenase metabolites as endogenous regulators of endothelial cell growth. These endothelial cells generate as their major lipoxygenase metabolite 15(S)-HETE, with a minor amount of 12(S)-HETE [165]. Application of either of these two lipoxygenase metabolites promoted significant DNA synthesis as well as proliferation of endothelial cells [165]. Very recently, the vital role of lipoxygenase products, 12(S)-HETE in particular, in con- trolling endothelial cell growth has been established in bovine capillary endothelial cells [166]. Beta FGF-, PDGF-, and serum-stimulated vascular cell (including both endothelial cells and smooth muscle cells) growth is dose-dependently inhibited by NDGA and a 12-1ipoxygenase inhibitor baicalein but not by indomethacin and a 5-1ipoxygenase inhibitor 5,6-dehydroarachidonic acid [166]. The growth of unstimulated endothelial cells was also inhibited by NDGA and baicalein [166], further lending support to the concept that 12(S)-HETE and perhaps some other lipoxygenase metabolites such as 15(S)-HETE are necessary growth signals for vascular cells. PDGF and bFGF are well-known angiogenic factors. The observations that their cell growth-promoting effects are to certain extent mediated through lipoxygenase metabolites such as 12(S)-HETE [166] strongly suggest that the latter by themselves may be important physiological angiogenic factors. In fact, 15(S)-HETE has been shown to enhance capillary endothelial cell migration at 0.1 gM and to be proangiogenic in an in vivo

rabbit corneal pocket assay (reviewed in ref. 165).

Whether 12(S)-HETE which is a structural isomer of 15(S)-HETE also shares similar proangiogenic properties is currently unknown. Considering that human stimulated blood can produce ~ 21 btM and 1 btM of 12(S)- and 15(S)-HETE, respectively [165] and that the levels of these metabolites can be further increased at the focal areas of tumor cell-platelet-endothelial cell interactions, it is reasonable to assume that these metabolites, especially 12(S)- HETE, may play a crucial role in modulating various phenotypic properties of endothelial cells such as adhesiveness, migration, and proliferation, as well as in their responses to growth factors and cytokines.

E The mechanisms o faction of l2(S)-HETE: a physiological PKC activator

12-HETE is the most abundant AA metabolite from human platelets [148] and is produced by platelets of all mammalian species so far examined [6]. As presented above, some tumor cells also express 12-1ipoxygenase mRNA, possess 12-1ipoxygenase enzyme, and generate 12-HETE. Arachidonic acid metabolites, including various lipoxygenase products, have been implicated in various signal transduction pathways involving growth factors, G-proteins, 1321 raS and ras-GAR cytokines, DAG and PKC, and neurotransmitters [149-158, 172, 173]. A growing body of recent evidence suggests that 12(S)-HETE may act as a second messenger in stim- ulus-response coupling in some cells [159-162]. For example, angiotensin II stimulates 12-HETE biosynthesis and aldosterone secretion by adrenal cortical cells; pharmacologic inhibitors of 12-HETE biosynthesis suppress angiotensin II-induced aldosterone secretion; and exogenous 12-HETE increases aldosterone secretion [160]. A similar series of observations suggest the participation of 12-HETE or its precursor in neurotransmitter peptide-induced hyperpolarization by Aplysia neuronal cells [159]. 12(S)-HETE, like LTB 4, has been shown to promote epidermal proliferation [161]. On the other hand, treatment of rat renal cortical slices with 12-HPETE or 12-HETE, but not LTB 4 or 5- HPETE, blocked the PGI 2- or iloprost-induced re-

381

nin secretion [163]. In addition, 12-HETE has been shown to significantly increase intracellular levels of cyclic AMP (cAMP) in human arterial smooth muscle cells [164].

Our laboratory has performed comprehensive studies on the molecular mechanisms underlying the versatile 12(S)-HETE effects on various tumor cells as well as vascular endothelial cells. Early experimental observations that 12(S)-HETE mimicked the tumor promoter PMA in enhancing tumor cell integrin expression and adhesion [108] lent credence to the postulate that 12(S)-HETE may work via activating PKC. Supportive evidence for this hypothesis was obtained when we observed that, in rat Walker carcinosarcoma (W256) cells, 12(S)-HETE induced a 100% increase in membrane-associated PKC activity and that down-regulation of PKC by prolonged treatment with PMA abolished the 12(S)-HETE enhanced W256 cell adhesion to endothelium [87]. Subsequently, it was observed that essentially all of the 12(S)-HETE evoked biological responses (in both tumor cells and endothelial cells) encompassing rearrangement of cytoskeleton, promotion of cell adhesion and spreading, induction of endothelial cell retraction, release of proteolytic enzymes, and enhance- ment of cell motility can be abrogated by selective PKC inhibitors [15-19, 87, 112, 116, 118, 119, 125,126, 135-137, 147]. Experiments by others also have shown that 12(S)-HETE and some other HETEs (such as 15-HETE) activate PKC [174]. PKC has been considered one of the most important phos- pholipid/Ca2+-dependent serine/threonine protein kinases localized in the center of various signaling pathways [167]. It is now known that PKC is a family of protein kinase which has at least 10 members, some of which are Ca2+-dependent (so-called con- ventional PKCs or cPKC) and some Ca2+-independ - ent (including novel PKCs or nPKC and atypical PKCs or aPKC). Different tissues and cells differentially express a specific repertoire of PKC isoforms that perform specialized functions. Although the precise role of PKC in multi-step tumorigenesis and cancer metastasis remains to be established, generally it is acknowledged that abnormal expression of specific PKC isoforms is closely associated

382

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1 2 3 4 5 6 7 8 9 10 11 12 13 14

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Fig. 5. Murine B16a melanoma cells express only the a isoform of PKC. Whole cell lysates were prepared from either cultured B16a cells (lanes 1, 3, 5, 7, 9, 11, and 13) or rat brains (lanes 2, 4, 6, 8, 10, 12, and 14). Equal amounts of proteins were loaded onto 10% denaturing SDS-PAGE and separated proteins transferred to nitrocellulose. The membrane was then probed with isoform-specific anti-PKC peptide antibodies (kindly provided by Dr. Y. Hannun, Duke University). The M,W. (in Kd) standards were shown on the left.

with cellular t ransformat ion and progress ion [168-

171]. The wel l -known physiological act ivators of P K C

are diacylglycerol ( D A G ) and Ca 2+. H o w 12(S)-

H E T E activates P K C has not been decisively de-

fined. It appears that 12 (S ) -HETE in different cell

types activates different isoform(s) of PKC. In murine B16a m e l a n o m a cells, PKCo~is the only i soform

that can be detec ted by immunoblo t t ing using iso-

form-specif ic ant i-pept ide antibodies (Fig. 5).

12 (S) -HETE st imulat ion leads to an increased

t ranslocat ion of P K C f rom the diffuse cytoplasmic

pool to the plasma m e m b r a n e (Fig. 6A and B).

W h e n B16a cells plated on f ibronect in were stim-

ulated with 12(S) -HETE, enhanced membrane-as - sociated P K C was observed to colocalize with vin-

A PKC B Vinculin

Fig. 7. B16a cells plated on fibronectin were stimulated with 0.1 gM of 12(S)-HETE for 15 min and then processed for double labeling of PKC (A) and vinculin (B). Bar, 10 gm.

culin to the focal adhesion plaques (Fig. 7). In rat AT2.1 prostate carcinoma cells, two isoforms of PKC, i.e., c~ and 5, were identified [118]. Interesting- ly, 12(S)-HETE preferentially activates PKCct by increasing the membrane association of this isoform, resulting in an upregulated tumor cell motility and invasion [118]. Treating tumor cells with thymelea toxin, which is a selective activator of PKCcz over Ca2+-independent PKCS, also promoted tumor cell motility and invasion [118; Fig. 8], thus con- firming the preferential activation of PKCc~ by 12(S)-HETE in this tumor cell line. Further, BAP- TA, a Ca2+-chelator, dose-dependently inhibited 12(S)-HETE-promoted tumor cell motility and invasion [118; Fig. 9], suggesting that the PKC isoform activated by 12(S)-HETE is Ca2+-dependent. In murine microvessel endothelial cells (i.e., CD3 cells), at least two isoforms of PKC are expressed (Liu, Tang, and Honn, unpublished observations). Which isoform of PKC or whether both isoforms of PKC are activated by 12(S)-HETE is not yet known. In rat adrenal glomerulosa cells, the c~ and ~ isoforms of PKC are expressed [175]. In sharp contrast, as described above, to the situation in tumor cells, 12(S)- HETE and 15(S)-HETE alike predominantly activate PKCe, although angiotensin II increases the membrane-bound levels of both PKCc~ and PKCe [175].

Exactly how 12(S)-HETE activates PKC is unclear. What is known is that this fatty acid does not directly activate PKC as assessed by PKC activity assays with 12(S)-HETE included in the reaction mixtures [193]. However, 12(S)-HETE stimulation of tumor cells is observed to be followed by a rapid accumulation of DAG and IP 3 [193], therefore suggesting PIP 2 metabolism through the action of PLC. To support this, blocking the functions of G proteins by pertussis toxin in B16a melanoma cells as well as inhibiting the PLC activity by specific antag- onists also suppress the effects of exogenous 12(S)- H E T E [193], indicating that 12(S)-HETE may function via activating an upstream G-protein. Another apparent alternative for the accumulation of DAG following 12(S)-HETE treatment is through down- regulating the DAG kinase activity, as proposed by Setty et al. [165]. In the phosphoinositide cycle, DAG derived from various sources undergoes rap-

383

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Fig. 6. 12(S)-HETE promotes PKCo~ association with plasma membrane, which is blocked by 13(S)-HODE. Immunofluores- cent labeling was performed using anti-PKCc~ as the primary antibody followed by incubating cells with FITC-labeled secondary antibody. In control (unstimulated) B16a cells, PKC takes the form of diffuse cytoplasmic staining (A). Stimulation of cells with 0.1 p.M of 12(S)-HETE (15' at 37 ° C) resulted in an increased association of PKC with the membrane (revealed as brightening dots in the picture, B). However, pretreatment of cells with 0.1/aM 13(S)-HODE (15" at 37 ° C) completely blocked the 12(S)-HETE effect (C). Bar, 10 btm.

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Fig. 8. PKCc~ activator thymelea toxin promotes motility and invasiveness of rat prostate carcinoma cells (AT2.1). In motility assays (A),

cells were treated with 0.01 or 0.1 btM thymelea toxin for 60 min. Cells in the control group were treated with vehicle ethanol (0.002%).

Data are f rom a representat ive exper iment and similar results were obtained in two other experiments. *: p < 0.001 compared to control; +: p < 0.005 compared to 0.01 gM thymelea toxin. B. To determine the effect of thymelea toxin on AT2.I cell invasion, cells were mixed with toxin containing medium to the indicated final concentrat ions and loaded into the invasion chambers. Data represent mean + SEM

of three exper iments performed in duplicate. #: p < 0.05 compared to control; @: p < 0.05 compared to 0.001 btM thymelea toxin. For

detailed experimental protocols, see ref. 118.

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Fig. 9. Effect of calcium chelator BAP T A on AT2.1 cell motility and invasion. In motility assays (A)~ cells were treated with indicated

concentrat ions of B A P T A / A M for 30 min or pretreated for 30 min followed by I2(S)-HETE (0.1 [aM) for 15 rain. Control cells were

treated with appropriate amount of vehicle DM S O (0.19 %). No difference in basal motility was observed when compared to cells treated with RPMI alone. Results are expressed as a percentage of the motility of control cells and are the mean + SEM of three separate

experiments. B. To determine the effect of B AP T A on invasion, AT2.1 cells (1 x 10 ~) were pretreated with indicated concentrat ion of

B A P T A / A M for l0 min, pelleted and resuspended in medium. Treated cells (5 x 104) were loaded into the invasion chambers and 12(S)~

H E T E or vehicle ethanol (0.03%) was added to the final concentrat ion of 0.1 ~aM. Data represent mean + SEM of four experiments.

id phosphorylation by DAG kinase to phosphatidic acid [176]. In bovine aorotic endothelial cells, 12(S)- HETE as well as 15(S)-HETE were found to inhibit the DAG kinase activity, leading to an accumulation of DAG mass in these cells [165]. Certainly it is possible that both mechanisms (i.e., the activation of upstream G-protein and PLC and the inhibition

of downstream DAG kinase) exist in cells resulting, eventually, in an increase in DAG and then activation of specific PKC isoform(s).

In various experimental settings we observed that the 12(S)-HETE effects (integrin expression, adhesion, spreading, retraction, etc) on tumor cells and vascular endothelial cells are antagonized by

386

13(S)-HODE 12(S)-HETE

P / T u r n o v e r - - - - - - -

Inactive PKC 1

Protein phosphorylation and activation of other kinases

Adhesion, spreading, motility

+ Metastasis

Fig. 10. A schematic illustration of the molecular mechanisms of the 12(S)-HETE effects. Detailed description and relevant references are given in the text.

13(S)-HODE, a bioactive lipid derived from linoleic acid metabolism by 15-1ipoxygenase [87, 99,108, 134, 146]. The 13(S)-HODE effects appear to be mediated through an inhibition of PKC activation by 12(S)-HETE. However, as in the case of 12(S)- HETE, 13(S)-HODE does not directly suppress the PKC activation [193]. In epidermis where 13- HODE is the major metabolite of linoleic acid, this fatty acid was found to be incorporated into 13- HODE-containing diacylglycerol, i.e., 13-HODE- DAG [177]. Interestingly, 13-HODE-DAG was demonstrated to selectively inhibit the PKC-[3 activity in epidermal cells [177]. Nevertheless, whether 13-HODE-DAG directly binds to PKC-[3 and subsequently inhibits its activity or 13-HODE-

DAG achieves the inhibition of PKC via some other signaling intermediates is unknown.

It is now well-appreciated that various cyclooxygenase products including prostaglandins, prostacyclin, and thromboxanes exert their functions through binding to specific cell surface receptors [22, 178]. So far many receptors and subtype receptors have been cloned and sequenced for these eicosanoids and most of them are shown to be typical G protein-linked receptors [22, 178-181]. Accumulat- ing experimental data suggest that 12(S)-HETE also may have a cognate cell surface receptor or binding site(s). Binding studies using human epidermal cell line SCL-II demonstrated that these cells possess a single class of binding sites for 12(S)-HETE with a Kd of 2.6 nM and a Bma X of 216,000 sites per

cell [182]. In human platelets, 12-HETE has previously been shown to block PGH2/TXA2-induced platelet aggregation [183]. Later, part of the inhibitory effect of 12(S)-HETE on PGH2/TXA2-induced platelet aggregation was found to be due to an inhibition of PGHR/TXA 2 binding to their com- mon receptor sites [184]. These observations suggest a possibility of 12(S)-HETE interaction with or binding to the PGH2/TXA z receptors. More recently, 12(S)-HETE binding sites have been reported in Lewis lung carcinoma (LLC) cells [185]. Intriguing- ly, subsequent subcellular fractionation studies by these authors identified potential 12(S)-HETE binding sites in multiple subcellular compartments [186]. Specifically, ~ 66.6, 38, 18, and 24 fmol/20 x 106 cells of 12(S)-HETE binding sites are identified for cytosol, mitochondria, plasma membrane, and nuclei, respectively [186]. The cytosolic binding sites for 12(S)-HETE appear to be a huge macro- molecular aggregate of an approximate M.W. of 669,000 dalton which is sensitive to trypsin or chy- motrypsin [186]. Our own studies also suggest that 12(S)-HETE binds to specific (receptor) sites in tumor cells as well as in endothelial cells [194].

Based upon the above discussions, we present the following working hypothesis to explain the involvement of 12(S)-HETE in regulating tumor metastasis, as illustrated in Fig. 10 and 11. External 12(S)- HETE, through binding to the cell surface receptor, activates a pertussis toxin-sensitive G protein [193], which in turn activates PLC. Stimulation of PLC activates the phosphoinositide metabolism generating from PIP 2 both DAG and IP3, which together activate PKC. Functionally activated PKC mediates protein (e.g., cytoskeletal elements) phosphorylation resulting in a modulation of a variety of phenotypic properties of metastasizing tumor cells such as adhesion, spreading, and motility (Fig. 10). 13(S)-HODE antagonizes the 12(S)-HETE effects by binding to either the same binding site(s) for 12(S)-HETE or different binding site(s) which neg- atively affect the function of 12(S)-HETE receptors (Fig. 10). In another scheme (Fig. 11), tumor cell adhesion to endothelial cells activates the 12-1ipoxyge- nase, leading to arachidonic acid metabolism to 12(S)-HETE. 12(S)-HETE, by unknown mechanisms, activates PKC and translocates PKC to the

387

plasma membrane. PKC subsequently phosphory- lares and reorganizes the cytoskeletal elements which mobilize cdlb133 integrin-containing vesicles to the plasma membrane. Cytoskeletal rearrangements induced by 12(S)-HETE also are involved in modulation of tumor cell spreading on matrix and tumor cell motility. Tumor cell-derived 12(S)- HETE likewise acts on endothelial cells, resulting in enhanced ~v[33 integrin surface expression and tumor cell adhesion, and PKC-dependent phosphorylation and reorganization of cytoskeleton and endothelial cell retraction. In addition, 12(S)-HETE may promote tumor cell release of cathepsin B to degrade the subendothelial matrix. In vivo, tumor cell interactions with platelets (through ~IIb[33 integrins and many other receptors) will lead to platelet aggregation, activation and release reactions. Platelet-derived 12(S)-HETE, which can be released to the loci of tumor cell-platelet-endothelial cell interactions, can similarly act on endothelial cells to initiate the diverse biological responses described above, thus contributing to tumor cell metastasis (Fig. 1l).

Perspectives

Multiple determinants have been implicated in tumor progression and metastasis. No single factor alone has been known to determine the fate of metastasizing tumor cells. 12-Lipoxygenase obviously is among those factors that facilitate the metastatic process. This enzyme is found to be expressed in many types of tumor cells in addition to platelets, leukocytes, epidermal cells, and some other normal cells. We have obtained preliminary evidence that the platelet-type 12-1ipoxygenase is amplified at the mRNA level in certain human cancers and there appears to be a positive correlation between the mRNA levels of 12-1ipoxygenase and the degree of malignancy [195]. Therefore, the possibility exists that 12-1ipoxygenase can be used clinically as a prognostic indicator for these cancers. Given the wide-spectrum modulatory role of 12(S)-HETE in tumor cell metastasis, functional 12-1ipoxygenase inhibitors such as baicalein and BHPP or their analogs may find applications in adjuvant therapies of

i ¸ ~

389

cancer patients aimed at interfering with or blocking metastasis. Likewise, prostacylin and 13(S)- HODE, which are known to antagonize many of the 12(S)-HETE effects, may also be useful in cancer treatment (see also chapter 11 for relevant discussions). Furthermore, studies have demonstrated that 12(S)-HETE could represent a crucial intermediate signaling molecule that mediates the functions of many growth factors and cytokines such as bFGF, PDGF, and AMF, etc. Thus, development of agents that directly interfere with or antagonize the functions of 12(S)-HETE may prove to be meaning- ful for the anti-signaling therapy of cancer metastasis, as currently advocated by many oncologists [187, 188].

Conclusion

- 12-Lipoxygenase catalyzes the transformation of AA to 12-HPETE which is then converted, either enzymatically or non-enzymatically, to 12(S)-HETE. There are at least three different types of mammalian 12-1ipoxygenases, which differ in their tissue distributions, preferential substrates, and primary products.

- The substrate specificity of 12-1ipoxygenases vs other lipoxygenases is determined by some well- conserved amino acid residues at or near the enzyme active center. The presence of multiple upstream activator sequences, in particular, an AP-2 site, in the promoter region of 12-1ipoxyge- nases provides the molecular basis for the induction of the enzyme in various mammalian cells.

- Many solid tumor cells express 12-1ipoxygenase mRNA and protein. 12-Lipoxygenases expressed in tumor cells are regulated by multiple extra- and intracellular factors. The ability of tumor cells to biosynthesize 12(S)-HETE is a positive correlate of their metastatic potential.

- 12(S)-HETE is a key intracellular signaling mol-

ecule that activates protein kinase C. Exogenous and tumor cell-derived 12(S)-HETE modulates a wide variety of properties of tumor cells (adhesion, spreading, invasion, motility, etc) and endothelial cells (retraction, proliferation, adhesion and migration, etc). 12-Lipoxygenase and 12(S)-HETE may represent key molecules to which anti-cancer and anti-metastatis therapeutic regimens are targeted.

Key unanswered questions

- How many different types of 12-1ipoxygenases are expressed by a distinct type of tumor? Are there any key differences between tumor cell expressed 12-1ipoxygenases and their counterparts in normal cells in terms of substrate specificity and profile of the metabolites?

- How does 12(S)-HETE exert its biological functions in tumor cells and endothelial cells? Are there authentic 12(S)-HETE binding sites or receptors? How does 12(S)-HETE activate protein kinase C?

- How can we exploit our knowledge about 12-1i- poxygenase and 12(S)-HETE to develop effica- cious anti-cancer and anti-metastasis protocols?

Acknowledgements

Original work in the authors' laboratories was sup- ported by National Institute of Health grants CA47115, CA 29997 (KVH), NATO Linkage Grant Programs LG92311 and the Fogarty International Research grant (KVH and JT) and grants from the Development Office of Harper Hospital and Wayne State University (KVH).

<

Fig. 1l. Role of 12-Iipoxygenase and 12(S)-HETE in tumor cell-vasculature interactions. Details are given in the text. TC, tumor cell; EC, endothelial cells, SEM, subendothelial matrix; 12-LOX, 12-1ipoxygenase; AA, arachidonic acid; CSK, cytoskeleton; PKC, protein kinase C; PGI2, prostacyclin. Modified after 98.

390

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Address for offprints: K.V. Honn, Division of Cancer Biology, Department of Radiation Oncology, Wayne State University, 431 Chemistry, Detroit, M148202, USA

12-Lipoxygenases and 12(S)-HETE: role in cancer metastasis

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