Arachidonic Acid Transformation and Tumor Production

A RA CHID0NIC AC ID TRANS FOR MATI 0N AND TUMOR PRODUCTION Lawrence Levine Department of Biochemistry. Brandeis University. Waltham, Massachusetts

I. Introduction ........................................................... 11. Arachidonic Acid Transformation ........................................ 111. Prostaglandin Levels in Tumors.. ....................................... IV. Arachidonic Acid Transformation and Hypercalcemia ..................... V. Arachidonic Acid Transfonnation and Tumor Promotion ................... A. Effects of TPA on Prostaglandin Production by Cells in Culture .................................................... B. Inhibitors of Prostaglandin Production: Their Effects on the Activities of TPA in Cells in Culture ................................. C. Effects of TPA on Prostaglandin Production in Vim ................... D. Stimulation of Prostaglandin Production by Growth Factors . . . . . . . . . . . VI. Prostaglandins: Their Effects on Cell or Tumor Growth ................... VII. Prostaglandins and the Immune Response ............................... VIII. Challenges ............................................................ References ............................................................

49 49 52 55 58 59 61 65 66 67 69 71 73

I. Introduction

Several observations have encouraged considerable speculation on the relationship between prostaglandins and cancer. Increased prostaglandin levels have been found in blood and/or urine of animals carrying neoplasms, as well as in transformed cells growing in tissue culture. In addition, it has been shown that prostaglandin production is associated with tumor promotion. Whether or not these associations are causally or casually related in such a complex of diseases is not clear. However, the concept that prostaglandins affect tumor growth is a constant theme in the mechanisms proposed to explain the phenomenon. Several reviews on the relationship of prostaglandins to cancer have been published (Bennett, 1979; Easty and Easty, 1976; Goodwin et al., 1980; Jaffe, 1974; Karim and Rao, 1976; Karmali, 1980; Pelus and Strausser, 1977). II. Arachidonic Acid Transformation

Some of the pathways of arachidonic acid metabolism are shown in Fig. 1. Arachidonic acid, which is the unsaturated fatty acid found in 49 ADVANCES IN CANCER RESEARCH, VOL. 35

Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-006635-1

HO LTA

Glulolhione S- Transferose

0

I

c::3

0

o--?-CooH

OOH

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

51

highest concentration in cellular phospholipids, is considered in detail; but other unsaturated fatty acids can also serve as substrates for the cyclooxygenase. The endoperoxides (PGG2 and PGH2) are the immediate products of the cyclooxygenase (Hamberg and Samuelsson, 1973; Nugteren and Hazelhof, 1973) and can undergo enzymatic and nonenzymatic transformations to form thromboxanes (TxA2and TxB2) (Hamberg et al., 1975; Needleman et al., 1976), prostacyclins (PG12) (Moncada and Vane, 1977), and prostaglandins (PGE2, PGFza, and PGD2). These endoperoxide metabolites can exist in classes depending on the degree of unsaturation in their precursor fatty acids; e.g., arachidonic acid (eicosatetraenoic acid) is transformed to class-two (PGEZ, PGFz,, etc.) products, and eicosatrienoic acid is transformed to class-one (PGE1, PGFla, etc.) products. The endoperoxides, and the products formed from the endoperoxides, are potent pharmacologically; and their activities, qualitatively and quantitatively, depend on the nature of the enzymatic (and nonenzymatic) transformations. Arachidonic acid also can be converted by lipoxygenases to hydroperoxyarachidonic acids and the corresponding hydroxyarachidonates (Hamberg and Samuelsson, 1973). Arachidonic acid is substrate for lipoxygenases, which also produce potent pharmacologically active compounds. The product of lipoxygenases acting at the 5 double bond of arachidonic acid can form a 5,6-epoxyarachidonic acid intermediate (leucotriene A). Leucotriene (LT)A can be transformed either to the potent chemotactic lipid 512-dihydroxyarachidonic acid (LTB) or, in the presence of glutathione and glutathione S-transferase, to the glutathione-containing compound (LTC). LTC, in some tissues, can be further metabolized b y y-glutamyl transpeptidase to form the cysteinyl-glycyl-containing compound (LTD). LTC, LTD, and the cysteinyl-containing compound (LTE) comprise a family of pharmacologically active compounds termed slow reactive substances of anaphylaxis (SRS-A) (Borgeat and Samuelsson, 1979;Jakschiket al., 1977; Morris et al., 1980a,b; Murphy et al., 1979; Orning et al., 1980; Parker et al., 1980). Prostaglandins, prostacyclins, and thromboxanes are not stored to any considerable extent in mammalian tissues. Any increase in their levels probably is brought about by a physiological stimulation and their rapid biosynthesis. This synthesis is often limited by the availability of precursor polyunsaturated fatty acids (Lands and Samuelsson, 1968; Vonkeman and van Dorp, 1968). Unsaturated fatty acids do not exist free in cells but are found in the form of phosphoglycerides and triglycerides, which must b e deacylated to provide substrate for the lipoxygenases and cyclooxygenases. The tissue phos-

52

LAWRENCE LEVINE

pholipids are the richest source of these precursor polyunsaturated fatty acids, and it has been postulated that the phospholipases of the cell are part of the sequence of events involved in prostaglandin biosynthesis (Kunze and Vogt, 1971). Deacylation of phospholipids probably occurs by more than one enzymatic pathway. For example, in platelets, three mechanisms of acylhydrolase activity have been proposed: (1) phosphatidylcholine and phosphatidylinositol are substrates for distinct phospholipase A, activities (Bills et al., 1977); (2) phosphatidylinositol is a substrate for sequential activities of phospholipase C (Rittenhouse-Simmons, 1979) and diacylglycerol lipase (Bell et al., 1979); ( 3 ) phosphatidic acid, generated by sequential actions of a phospholipase C on phosphatidylinositol and phosphorylation of the diacylglycerol, stimulates the phospholipase A, attack of phosphatidylcholine (Lapetina et al., 1980). In rat mast cells, a pool of phosphatidylcholine, synthesized from phosphatidylethanolamine by three methylation steps, is attacked by phospholipase A, (Hirata and Axelrod, 1980); in methylcholanthrene-transformed mouse fibroblasts, as a result of stimulation by bradykinin, phosphatidylinositol is deacylated by phospholipase A,; phosphatidylcholine also may be hydrolyzed by phospholipase A, (Hong and Deykin, 1979, 1981). Ill. Prostaglandin Levels in Tumors

Most mammalian cells have the enzyme(s) that synthesize endoperoxides from arachidonic acid. The capacity to generate thromboxanes, prostacyclin, and the prostaglandins from endogenous endoperoxides varies considerably among cells (Levine et al., 1979). For example, in endothelial cells derived from bovine aorta, more than 90% of cyclooxygenase products is PGI,, approximately 80% of the products of endothelial cells derived from bovine adrenal is PGI,, whereas only 26% of the cyclooxygenase metabolites derived from human umbilicus vein endothelial cells is PGI,. Marcus et al. (1978) found that 50% of cyclooxygenase products of endothelial cells derived from umbilical cords is prostacyclin. Cells derived from murine lymphoma cells (WEHI) synthesize relatively large amounts of thromboxane (around 60% of all cyclooxygenase products). PGE, (16%), PGF, plus PGF,, (19%), and 6-keto-PGF1, (about 4%) were also found. This cell line is a macrophage-like cell. Guinea pig macrophages have been reported to produce in culture PGE, (Gordon et al., 1976); and mouse macrophages, PGE, and 6-keto-PGF,, (Humes et al., 1977) and PGE, and

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

53

thromboxane (Brune et al., 1978a). Subsets of monocytes and macrophages appear to be capable of metabolizing arachidonic acid differently. Around 65% of cyclooxygenase products synthesized by WI-38 is PGEZ. Thromboxane (12%), PGF2, (18%), and prostacyclin (3%) are other biosynthetic products. D-550 cells, a fibroblast cell line derived from normal human foreskin, synthesizes mainly PGE, and PGF,,, in a ratio of 4 to 1, respectively. Similar percentages of PGE2 and PGF2, are produced by the adult type I1 alveolar cells isolated from rat lung. Taylor et al. (1979) report that these adult type I1 alveolar cells synthesize mainly PG12: 80% of cyclooxygenase products measured by release of radiolabeled compounds from [SH]arachidonic acid-prelabeled cells was found to be 6-keto-PGF1, . About 70% of the cyclooxygenase products of epithelial-like dog kidney cells is PGF2,. This cell also synthesizes considerable amounts of prostacyclin (22%) and PGE2 (9%).The cyclooxygenase products synthesized by some cells in culture are shown in Table I. Thus, it is not surprising that most extracts of tumors contain cyclooxygenase products. In most of the early studies on prostaglandin content of tumors, only PGE2and PGFzawere measured. The levels of these prostaglandins in tumors or the tumors’ biosynthetic capacities usually were compared to those of the appropriate normal tissue. PGE, and/or PGFza have been found in extracts of medullary, anaplastic and papillary carcinomas of the thyroid (Jaffe and Condon, 1976; Kaplan et al., 1973; Williams et al., 1968), neuroblastoma (Williams et al., 1968), pheochromocytoma (Papanicolaou et al., 1975; Sandler et al., 1968), islet cell tumors (Sandler et al., 1968), colonic carcinoma (Bennett et al., 1977a),breast carcinoma (Bennett et al., 1977b, 1979, 1980b; Rolland et al., 1980; Stamford et al., 1980),bronchial carcinoma (Fiedler et al., 1980), renal cell carcinoma (Zusman et al., 1974), and in several other neoplasms (Goodwin et al., 1980; Husby et al., 1977). Prostaglandins have been found in extracts of experimental tumors. PGE2, as measured by bioassay and identified by gas chromatographic and mass-spectrometric analysis, is present in BP8/ P, ascites cells and in BP8/P1 solid tumors of mice as well as in the mouse sarcoma 180 tumors (Sykes and Maddox, 1972). A large increase in PGE and PGF is found in virus-induced Maloney sarcoma tumors when compared with the levels of those prostaglandins in the normal leg muscles of these mice (Humes and Strausser, 1974; Humes et al., 1974; Strausser and Humes, 1975). We have found that extracts of a mouse fibrosarcoma, HSDM1, contain large amounts of PGE2 (Tashjian et al., 1972), as does the VX, carcinoma of rabbit (Voelkel et al., 1975).

ARACHIWNIC

Cell line

Source

TABLE I ACID METABOLISM B Y CELLS IN CULTURE"

Stimulant

Analysis technique

% Cyclooxygenase product in culture fluid

&Keto-PGF,, ______

WEHI-5 Endothelial MDCK WI-38

D-550

RBL- 1

Mouse lymphoma Bovine aorta Dog kidney Normal human embryonic lung Normal human foreskin Mouse leukemia

Melittin (1 &ml) Melittin (1 pg/ml) TPA (1 ng/ml) Melittin (1&ml) Melittin (1pg/ml) A-23187 (1pg/ml)

RIA RIA RIA RIA RIA RIA

4

93 22 3 1

1

TxBI

PGF2,

PGE,

PGD,

58 1 <1 12 1 0

19 3 68 18 19 22

16 2 9 67 79

1 <1 <1 <1 (1 16

~~

60

a The serologic specificities of the appropriate anti-arachidonic acid metabolites have been reported in the following: 6-Keto-PGF1,, Gaudet et al. (1980);TxB,, Alam et al. (1979);PCFZ,, Levine et al. (1971),Pong and Levine (1977);PCEI, Levine et al. (1971), Pong and Levine (1977);PGDz, Gaudet et al. (1980), Levine et al. (1979).

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

55

Possibly of more significance in assessing the relationship of arachidonic acid transformation to cancer are the findings of elevated prostaglandins in the blood and urines of patients with cancers or in animals bearing tumors. Bennett (1979) has extensively reviewed the literature dealing with levels of prostaglandin found in the blood of patients with tumors. Increases have been found in the blood of patients with medullary carcinoma of the thyroid (Jaffe and Condon, 1976; Kaplan et al., 1973; Williams et al., 1968), neuroblastoma (Williams et al., 1968), Kaposi’s sarcoma (Bhana et al., 1971), breast cancer (Bockman and Myers, 1977; Powles et al., 1977; Rolland et al., 1980; Stamford et al., 1980), renal cell carcinoma (Cummings and Robertson, 1977), bronchial carcinoma (Fiedler et al., 1980), and a variety of other cancers (Demers et al., 1977, 1979; Demers and Derck, 1980). Increases in prostaglandin metabolites are present in urines of a variety of patients with cancer (Seyberth e t a l , 1975).

IV. Arachidonic Acid Transformation and Hypercalcemia

In a significant number of patients with cancer, the accompanying hypercalcemia appears to have a humoral etiology; resection of the tumors in such cases leads to remission of the hypercalcemia, and reappearance of the tumor is accompanied by the hypercalcemia (Tashjian, 1978). Production by the tumors of serologically active parathyroid hormone has been implicated, but parathyroid hormone is found only in a minority of such patients (Powell et al., 1973). Some arachidonic acid transformation products are potent bonereabsorbing substances in vitro (Bennett et d., 1980a; Dietrich e t d . , 1975; Klein and Raisz, 1970; Raisz et al., 1977; Santoro et al., 1977; Tashjian et al., 1977~). PGE, is the most potent of the cyclooxygenase metabolites, but other products and their metabolites also possess bone-resorbing activities. The bone-resorbing activities of many of the lipoxygenase products are not known. Constant intravenous infusion of exogenous PGE, in the unanesthetized, intact rat leads to elevated plasma calcium levels (Franklin and Tashjian, 1975), but infusion of PGEz or PGEl into the thoracic aorta does not affect plasma calcium levels in the dog (Belie1 et al., 1973). Intraarterial PGE, infusion in thyroparathyroidectomized, but not intact, rats elevates plasma calcium levels (Robertson and Baylink, 1977). IntraperitoneaI injections of 16,16-dimethyl-PGE,-methylester,a long-acting synthetic analog of PGE,, result in enhanced bone resorption as measured histologically (Santoro e t aZ., 1977). The humoral mediator of hypercalcemia

56

LAWRENCE LEVINE

was suggested to be PGE, (Tashjian et al., 1972) when it was found that a transplantable mouse fibrosarcoma HSDMl produced elevated plasma calcium concentrations two weeks after subcutaneous or intramuscular implantation of the tumor. This tumor does not invade bone. The tumor, as well as clonal strains of HSDMl cells grown in culture, produces large amounts of PGE,, which was identified as the bone-resorbing factor (Levine et al., 1972; Tashjian et al., 1974). Indomethacin, a potent inhibitor of cyclooxygenase activity, 5,8,11,14eicosatetraynoic acid, an inhibitor of both cyclooxygenase and lipoxygenase activities, and hydrocortisone, which blocks both cyclooxygenase and lipoxygenase pathways by preventing formation of substrate, inhibit the formation of PGE, by the cells in culture (Levine et al., 1972; Tashjian et al., 1972, 1974). In uivo, plasma levels of PGE,, and more strikingly its more stable metabolite 13,14-dihydro15-keto-PGE,, are elevated after tumor implantation (Tashjian et al., 1973, 1977a). The elevation of the plasma levels of the metabolite precedes that of the plasma calcium. Treatment of the tumor-bearing mice with indomethacin (Tashjian et al., 1973) or hydrocortisone (Tashjian et al., 1977a) prevents the elevation of both plasma 13,14dihydr0-15-keto-PGE~and plasma calcium, both of which are again elevated upon withdrawal of the indomethacin or hydrocortisone treatment. A second animal model in which the elevated levels of plasma calcium are associated with PGE, production by the tumor is the VX, squamous-cell carcinoma in the rabbit (Voelkel et al., 1975). In this tumor, as with HSDM, fibrosarcoma of the mouse, clonal strains of cells in culture as well as the tumor produce large amounts of PGE,. Both the PGE, and the bone-resorbing activities of the cells and tumors are inhibited by indomethacin. Rabbits have elevated plasma levels of 13,14-dihydro-15-keto-PGE, which precede the elevation of plasma calcium (Alam et al., 1980; Seyberth et al., 1977; Tashjian et al., 1977b) if the tumor is implanted intramuscularly. If, however, the tumor is implanted intra-abdominally, the plasma level of 13,14-dihydro-15-keto-PGEzis again elevated, but not that of plasma calcium (Hubbard et al., 1980).Treatment of the rabbits with indomethacin or hydrocortisone either prevents or reduces the hypercalcemia and elevated levels of 13,14-dihydro-15-keto-PGE,,depending on the time between the treatment and the intramuscular implantation (Tashjian et al., 1977b). The effects of such treatments are reversible. Whereas the elevation in peripheral venous plasma PGE, is difficult to detect, the elevation of its longer lived metabolites is striking. However, the level of PGE, in the venous drainage of the

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

57

V X 2 tumor is higher than that found in systemic venous plasma and that of the opposite femoral vein (Voelkel et al., 1975). The plasma concentrations of two acute-phase reactants, ceruloplasmin and haptoglobin, rise rapidly following implantation of the VX2 tumor (Voelkel et al., 1978). Plasma concentrations of ceruloplasmin rise in parallel with 13,14-dihydro-15-keto-PGEe, and both precede the rise in plasma calcium. Indomethacin prevents the rise in the ceruloplasmin and reduces the elevation once it has occurred. No changes in plasma albumin concentration are noted. It was suggested that arachidonic acid metabolites may play a role in the elevation of these acute-phase proteins in certain patients with malignant tumors, as well as in patients with certain chronic inflammatory diseases (Voelkel et al., 1978). Conditioned media from synovial tissue from patients with rheumatoid arthritis contain bone resorption-stimulating activity. The synovial cultures also produce PGE2.The production of both the bone resorption-stimulating activity and PGE, is inhibited more than 90% by treatment of the synovial cultures with indomethacin. It was suggested that the PGEz produced by rheumatoid synovia may contribute to the destruction of juxta-articular bone in rheumatoid arthritis (Robinson et al., 1975). Bone itself can metabolize arachidonic acid. PGE2 is produced by bone by a complement-mediated reaction (Raisz et al., 1974; Sandberg et al., 1977)as well as after treatment with crude collagenase (Dowsett et al., 1 9 7 6 ~ )Bone . is stimulated to transform arachidonic acid (PGE2 was measured) after stimulation b y 12-0-tetradecanoyl phorbol-13acetate (TPA), a potent tumor promoter, and melittin, a polypeptide isolated from bee venom (Tashjian et al., 1978), as well as epidermal growth factor (Tashjian and Levine, 1978). It was suggested that PGEz could act locally to stimulate bone resorption. The tumor promoters, epidermal growth factor, and melittin stimulate acylhydrolase activity of cells in culture (Hassid and Levine, 1977; Levine and Hassid, 1977a,b), so it is not surprising that arachidonic acid metabolites other than PGE2 are found in bone-culture-conditioned media. Fetal rat bones synthesize PGE2, PGF2,, and PG12 (Raisz et al., 1979). Mouse calvaria also synthesize PGE,, PGF2,, and PG12 and, in addition, oxidize the 15-hydroxy function of these prostaglandins to their respective 15-keto metabolites and reduce the 13,14 double bond to the 13,14-dihydro-15-keto compounds (Voelkel et al., 1980). When incubated with exogenous arachidonic acid, bone synthesizes thromboxane. PG12 stimulates bone resorption of fetal rat bones (Raisz et al.,

58

LAWRENCE LEVINE

1979), but conflicting data have been reported on the activity of PG12 on mouse calvaria (Bennett et al., 1980a; Voelkel e t al., 1980). However, in the VX2 squamous-cell carcinoma of the rabbit, elevations of PGIz in plasma, measured as its more stable product 6-keto-PGF1,, have not been found (Alam et al., 1979). It must be recalled that the bone-resorbing activities of the lipoxygenase products of arachidonic acid transformation have not been reported. The mouse HSDMl fibrosarcoma and the rabbit VX2 squamous-cell carcinoma could be visualized as animal models for tumors that mediate hypercalcemia in human malignancy. This concept is supported by the findings that indomethacin treatment reduces the hypercalcemia of a patient with a renal cell adenocarcinoma (Brereton et al., 1974) and that, in a patient with renal cell carcinoma, the hypercalcemia is accompanied by elevated plasma immunoreactive PGEr but low plasma parathyroid hormone (Robertson et al., 1975). Similar responses to indomethacin treatment, or the findings of elevated plasma levels of prostaglandins, in patients with hypercalcemias associated with a variety of malignancies have been reported (Demers et d., 1977; Seyberth et aZ., 1975). However, many patients with hypercalcemias associated with various malignancies do not respond to indomethacin treatment, nor can an elevated prostaglandin synthesis be demonstrated (Seyberth et aZ., 1978). V. Arachidonic Acid Transformation and Tumor Promotion

Carcinogenesis may be a multistep process (Boutwell, 1974), and it is probable that various environmental factors act at different steps in this process. In one of the best-studied models, the “two-stage carcinogenesis” system in mouse skin (Berenblum, 1969), two distinct stages designated “initiation” and “promotion” have been identified. Additional stages and cofactors may play a role in cancer induction in other tissues and species. The interaction of multiple factors probably is required for the induction of many human cancers. The essential features of the two-stage carcinogenesis model are the following: (1)one application of the initiator alone to mouse skin will produce few or no tumors; (2) many applications of the promoting substance alone will produce inflammation followed by epithelial hyperplasia but will not produce tumors; and (3) one application of the initiator followed by many applications of the promoter results in both benign and malignant tumors. The promoting stage has been separated into at least two stages (Boutwell, 1974). Tumor promoters and

AFIACHIDONIC ACID TRANSFORMATION AND TUMORS

59

mechanisms of tumor promotion have recently been reviewed (Diamond et al., 1980).

A. EFFECTSOF TPA ON PROSTAGLANDIN PRODUCTION BY CELLSIN CULTURE In the simple two-stage model in mouse skin (Berenblum, 1969), croton oil prepared from the seeds of Croton tiglium was the promoting agent. The fractions from the croton oil responsible for the activity have been purified and characterized by Hecker (1968) and van Duuren (1969) and their colleagues. Indeed, the most extensively studied tumor promoters are the phorbol diesters purified from croton oil. Among these, TPA is the most active. TPA stimulates several types of cells to produce prostaglandins (Table 11). The most responsive of these cells is the canine kidney cell line MDCK; as little as to loTLoM TPA is sufficient to stimulate prostaglandin production (Levine and Hassid, 1977b). Phorbol-12,13-didecanoate(PDD) at M and 10-lo M also stimulates prostaglandin production by MDCK cells, but the non-tumor-producing phorbol diester 4a-phorbol-12,13didecanoate (4a-PDD) is inactive even at lo-' M . One of the several activities of TPA on cells is deacylation of cellular

TABLE I1 CELLS IN WHICH AFUCHIDONIC ACID TRANSFORMATION IS STIMULATED BY TPA Cell

Source

Reference

M DCK WEHI-5 HSDMl LC-540 Smooth muscle WI-38 D-550 MC5-5 Macrophages Osteosarcoma

Dog kidney Mouse lymphoma Mouse fibrosarcoma Rat Leydig Bovine aorta Human embryonic lung Human foreskin Transformed mouse fibroblast Mouse peritoneum Human bone

Y-1

Mouse adrenal Mouse epidermis Chick embryo

Levine and Hassid, 1977b Levine et al., 1979 L. Levine, unpublished data L. Levine, unpublished data L. Levine, unpublished data Levine et al., 1979 Levine et al., 1979 Levine et al., 1979 Brune et al., 1978a M. A. Shupnik and A. H. Tashjian, Jr., unpublished data L. Levine, unpublished data Fiirstenberger et al., 1980 Mufson et al., 1979 Crutchley et al , 1980

HEU30 Fibroblasts HeLa

60

LAWRENCE LEVINE

phospholipids (Levine and Hassid, 197713; Ohuchi and Levine, 1978a). TPA and PDD, but not 4a-PDD, stimulate the release of arachidonic acid from the membrane phospholipids of MDCK cells. The hee arachidonic acid can then be metabolized by the cyclooxygenase system to the prostaglandins. TPA and PDD, but not 4aPDD, also alter the morphology of MDCK cells. These effects of TPA, deacylation of phospholipids, prostaglandin production, and alteration of cell morphology, require time for expression. Cycloheximide inhibits all three effects (Ohuchi and Levine, 1978a). Indomethacin inhibits the stimulated prostaglandin synthesis and, at high concentrations, also inhibits TPA’s effect on cell morphology (Levine, 1981). Indomethacin is a potent inhibitor of cyclooxygenase (Lands and Rome, 1976; Smith and Lands, 1971) but is also an inhibitor of phospholipase A2 (Kaplan et al., 1978), phosphodiesterase (Flores and Sharp, 1972), PGE2-9-keto reductase (Hassid and Levine, 1977b; Stone and Hart, 1975), and a CAMP-dependent protein kinase (Kantor and Hampton, 1979); it also inhibits some deacylation of cellular lipids (Ohuchi and Levine, 1978b). Unstimulated macrophages obtained from the peritoneal cavity of mice respond to TPA ( 10-s-lO-s M) and PDD (10”-10-* M) treatment by releasing PGE2 (Brune et al., 1978a). TPA (10-7-10-s M ) rapidly releases arachiodonic acid, PGE2, and PGFZafrom chick embryo fibroblasts, PDD, phorbol- 12,lSdibenzoate and mezerein are also active, whereas phorbol and 4a-PDD are ineffective (Mufson et al., 1979). Both the release of arachidonic acid and the stimulation of prostaglandin production are inhibited by cycloheximide and puromycin. Indomethacin inhibits TPA-induced prostaglandin synthesis but slightly enhances arachidonic acid release. TPA stimulates PGE2 and PGF,, production in HeLa cells (Crutchley et al., 1980), PGE2 biosynthesis in the murine epidermal cell line HEW30 (Fiirstenbergeret al., 1980), and production of PGE2, Fa,, and I2 by bone (Voelkel et al., 1980). TPA stimulates prostaglandin biosynthesis by activating acylhydrolases (Levine, 1979b; Levine and Alam, 1981; Levine and Hassid, 1977b; Levine and Ohuchi, 1978b; Ohuchi and Levine, 1978a). As outlined in Section 11, several biochemical pathways have been recognized within this deacylation reaction. There is selectivity among the acylhydrolases for substrate or enzyme, or both when TPA stimulates MDCK cells; MDCK cells in which the phospholipids are labeled with [‘4C]linoleic acid and [3H]arachidonic acid are stimulated by TPA to release arachidonic acid but not linoleic acid (Ohuchi and Levine, 197813).

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

61

B. INHIBITORS OF PROSTAGLANDIN PRODUCTION: THEIREFFECTS ON THE ACTIVITIES O F TPA IN CELLS IN CULTURE Indomethacin is one of the most potent inhibitors of prostaglandin production (Robinson and Vane, 1974). It prevents formation of endoperoxides by blocking cyclooxygenase activity (Lands and Rome, 1976; Smith and Lands, 1971). However, indomethacin also inhibits phospholipase A2 (Kaplan et al., 1978), acylhydrolase activity in MDCK cells (Ohuchi and Levine, 1978b), phosphodiesterase (Flores and Sharp, 1972), PGE2-9-keto reductase (Hassid and Levine, 1977b; Stone and Hart, 1975), and a CAMP-dependent protein kinase (Kantor and Hampton, 1979). In view of this nonspecificity, the effects of indomethacin may not be attributed solely to inhibition of prostaglandin biosynthesis (Flower, 1974). Indomethacin does inhibit TPAstimulated prostaglandin production by cells in culture. Another series of inhibitors of prostaglandin production are the anti-inflammatory steroids (Gryglewski et al., 1975; Kantrowitz et al., 1975; Lewis and Piper, 1975; Tashjian et al., 1975). In some cells, the corticosteroids have been shown to inhibit prostaglandin production by blocking the expression of acylhydrolase activity (Hong and Levine, 1976; Tam et al., 1977). The glucocorticoids may inhibit acylhydrolase activity by inducing biosynthesis of a phospholipase A2 inhibitor (Blackwell et al., 1980; Flower and Blackwell, 1979; Nijkamp et al., 1976). In rabbit neutrophils, glucocorticoids induce a phospholipase A2 inhibiting protein (Hirata et al., 1980). The mechanism of action of glucocorticoids may vary among cells and tissues. In 3T3 cells, corticosteroids stimulate cyclooxygenase activity, although arachidonic acid release is inhibited under some conditions (Chandrabose et al., 1978). In rheumatoid synovia, release of arachidonic acid is unaffected even under conditions where prostaglandin synthesis is more than 80% inhibited (Robinson et al., 1980). Preincubation of dexamethasone with smooth muscle cells prepared from bovine aorta inhibits the stimulation of prostaglandin production of TPA (L. Levine, unpublished data). The retinoids are another class of inhibitors that block tumor promotion. They are effective both in vitro and in uivo. Retinoids inhibit promoter-induced ornithine decarboxylase (ODC) activity in mouse epidermis (Verma and Boutwell, 1977; Verma et al., 1978). Those retinoids that inhibit TPA-induced ODC activity in mouse epidermis also inhibit TPA-induced ODC activity in phytohemagglutinin-treated bovine lymphocytes (Kensler et al., 1978) and TPA’s comitogenic activity in these lectin-treated cells (Kensler and Mueller, 1978). An-

62

LAWRENCE LEVINE

other inhibitor of TPA’s comitogenic effect on these cells is 5,8,11,14eicosatetraynoic acid (Wertz and Mueller, 1980), an inhibitor of lipoxygenase as well as the prostaglandin-producing cyclooxygenase. Retinoids, at low concentrations, stimulate plasminogen-activator synthesis in chick embryo fibroblasts, and at suboptimal levels of TPA the effects of retinoic acid and TPA are synergistic (Wilson and Reich, 1978). In MDCK cells the retinoids, cis-retinoic acid, trans-retinoic acid, retinyl acetate, retinol, retinal, retinyl palmitate, and trimethylme thoxyphenyl retinoic acid, do not affect deacylation of phospholipids or prostaglandin production, and at relatively high concentrations they even enhance them (Levine and Ohuchi, 1978a). In chick embryo fibroblasts, trans-retinoic acid ( 10-5-10-6 M ) inhibits TPA-induced arachidonic acid release and prostaglandin production (Mufson et al., 1979). The synthetic retinoid N-(4-hydroxyphenyl)retinamide,at levels ranging from 0.025 to 3.1 p M , inhibits serum- and TPA-stimulated biosynthesis of PGFz., 6-keto-PGF1., and PGEz by MDCK cells (Levine, 1979a). N-(4-hydroxyphenyl)retinamide also is a potent inhibitor of PGEz and PGFzu production by serum-stimulated methylcholanthrene-transformed mouse fibroblasts and normal human fibroblasts. In the presence of 10% fetal bovine serum, N (4-hydroxypheny1)retinamideinhibits prostaglandin production by MDCK cells 4 times less effectively than indomethacin and about 50 times more effectively than aspirin. However, N-(4-hydroxyphenyl)retinamide does not affect the release of radiolabeled materials from TPA-stimulated labeled cells; cyclooxygenase activity appears to be inhibited, but not acylhydrolases (Levine, 1979a). The contributions of the 4-hydroxy- and phenol- functions of N-(4-hydroxyphenyl)retinamide to its prostaglandin-inhibiting effectiveness can be seen in Fig. 2 (L. Levine and M. Sporn, unpublished data). Another inhibitor of TPA-stimulated prostaglandin production in MDCK cells is a-tocopherol (Ohuchi and Levine, 1980). a-Tocopherol inhibits prostaglandin production in TPA-stimulated MDCK cells but not in control cells. This inhibitory effect is observed only if the cells are treated with TPA in the presence of a-tocopherol. a-Tocopherol decreases TPA-stimulated prostaglandin production by inhibiting the binding of [3H]TPA to the cells. The mechanism is not clear since relatively large concentrations ( 102-103mole excess of a-tocopherol to TPA) are required to inhibit binding to the cells. It is possible that TPA is more soluble in a-tocopherol bound in the MDCK cell membrane or in an a-tocopherol micelle. Another possibility is that a receptor site for TPA is being reversibly blocked by a-tocopherol. There appears to be

Retinoid

@

A

A

.

L

O

”

Effect

-

p

on Prostaglondln Production

It

N-(Z-Hydroxyphenyl)retinornide

N- (3-HydroxyphenyNretinomide

N- Phenylret inomide

~ - ( 4 - ~ t h o x y p h e n Iratinornide yl

@

L

b

N

q

-

;

:

~-(4-Hydroxy-3-carboxypheny1)retinomide

N-(3-Methyl-4-hydroxyphenyl lrelinomide

.1 = strong inhibitor; + = no effect; t t = stimulation; = slight inhibition; f = slight stimulation (L.Levine and M. Sporn, unpublished data). FIG.2. Effects of some synthetic retinoids on prostaglandin production.

64

LAWRENCE LEVINE

some specificity for tumor promoters originating from Croton tiglium L.; a-tocopherol inhibits the effects of the tumor-promoting phorbol esters obtained from this plant but not the effects of diterpenoid esters isolated from the plant genera Daphne or Gnidia. TPA stimulation of prostaglandin synthesis requires protein synthesis. Cycloheximide from 0.05 to 0.5 pg/ml inhibits TPA-stimulated prostaglandin production and deacylation of cellular phospholipids in MDCK cells (Ohuchi and Levine, 1978a). Cycloheximide, 40 pg/ml, and puromycin, 20 pg/ml, inhibit TPA-stimulated prostaglandin production and acylhydrolase activity by more than 90% in chick embryo fibroblasts (Mufson et al., 1979). Inhibition of TPA-stimulated deacylation of cellular lipids and prostaglandin production b y indomethacin, dexamethasone, retinoic acid, N-(4-hydroxyphenyl)retinamide,a-tocopherol, and cycloheximide is summarized in Table 1x1. In MDCK cells, retinoic acid does not alter any of these responses, and at high concentrations it is synergistic with TPA (Levine and Ohuchi, 1978a). Indomethacin inhibits prostaglandin production very effectively, but it also inhibits the deacylation of cellular lipids (Ohuchi and Levine, 1978b). N(4-Hydroxypheny1)retinamideinhibits TPA-stimulated prostaglandin production but does not affect deacylation of lipids (Levine, 1979a). a-Tocopherol inhibits both effects, prostaglandin production and deacylation of cellular lipids; it does this by blocking the binding of TPA to MDCK cells. Dexamethasone, which inhibits prostaglandin

TABLE I11 DEACYLATION OF PHOSPHOLIPIDS AND INHIBITION OF TPA-STIMULATED PROSTAGLANDIN PRODUCTION BY CELLSIN CULTURE Inhibitor Cyc!oheximide" Indomethacin Retinoic acid' N-(4-Hydroxypheny1)retinamide a-TocopherolC Dexamethasoned

0.1 pg/ml. * 5 x 10-5 M (+); 1 x 10-5 M (-). ' MDCK cells. MC5-5 and smooth muscle cells.

Phospholipid deacylation

Prostaglandin production

+

+ + + + +

20

-

+ +

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

65

production in several cells by preventing the expression of acylhydrolase activity, inhibits TPA-stimulated prostaglandin production in smooth muscle cells (L. Levine, unpublished data). Cycloheximide (0.1 vg/ml) inhibits both TPA-stimulated reactions, most likely by inhibiting synthesis of a protein that stimulates prostaglandin production and acylhydrolase activity. Whereas the TPA-stimulated prostaglandin biosynthesis is inhibited by cycloheximide of 0.1 pg/ml, that of most other stimulators is not (Levine, 1981). However, cycloheximide at 2.0 pdml inhibits serum-, thrombin-, and bradykinin-stimulated prostaglandin synthesis by MC5-5 cells (Pong et al., 1977). C. EFFECTSOF TPA ON PROSTAGLANDIN PRODUCTION in Vivo There is good correlation between the irritant and promoting activities in mouse skin and PGE2-release in mouse macrophages in a series of diterpine derivatives (Brune et al., 197813). One of the pleiotypic effects of TPA on mouse skin is induction of ornithine decarboxylase (ODC) activity (Diamond et al., 1980). Indomethacin inhibits TPA’s induction of ODC in mouse skin (Verma et al., 1980). Moreover, this inhibition is completely blocked by treatment with PGEl and PGE2,but not by PGF1, and PGF,,. Indomethacin administered one hour before TPA completely inhibits the proliferative response of mouse epidermis to TPA; this inhibition is reversed by applying PGE2,but not PGFz., simultaneously with TPA (Furstenberger and Marks, 1978). TPA-induced skin inflammation is not influenced by indomethacin, suggesting that PGE, (or a closely related compound) mediates the mitogenic effect of TPA in mouse skin. TPA does not compete for hypothetical PGE receptors, at least not on adipose cells (Furstenberger and Marks, 1979a) as suggested by Smythies et al. (1975). Lupulescu (1978) has shown that mice treated with 3-methylcholanthrene and injected concomitantly with 10 pg of PGE, three times weekly for 2 months have increased skin tumors. When PGF, replaces PGE, in the intramuscular injections, no increase is observed. Two peaks of PGE biosynthesis, one at 10 minutes and a second at 60 minutes, are found in mouse skin following a topical application of TPA (Fiirstenberger and Marks, 1980). Pretreatment of the skin with indomethacin abolishes the 10-minute peak of PGE synthesis as well as the proliferative response induced by TPA. However, if the indomethacin is applied 30-60 minutes after application of the TPA, inhibition of the proliferative response is not observed. Thus, the early PGE synthesis was suggested to be an obligatory event for epidermal

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cell proliferation induced b y TPA. Another effective stimulator of prostaglandin production is the calcium ionophore antibiotic A-23187 from Streptomyces chartreusis (Gemsa et al., 1979; Knapp et al., 1977; Marks et al., 1981; Pickett et al., 1977; Rittenhouse-Simmons and Deykin, 1977; Waelbroeck and Boeynaens, 1977; Wantzell and Epand, 1978; Weksler et al., 1978). Topical application of A-23187 on mouse skin induces in vivo prostaglandin-mediated epidermal hyperplasia and inflammation similar to those initiated by TPA (Marks et al., 1981). However, A-23187 does not have tumor-promoting activity. Thus, these two biological effects of TPA, prostaglandin-mediated epidermal hyperplasia and inflammation, are not indicative of tumor-promoting capacity (Marks et al., 1981). Natural and synthetic glucocorticoids inhibit tumor promotion (Belman and Troll, 1972; Ghadially and Green, 1954; Schwarz et al., 1977). Topical application of dexamethasone inhibits TPA-mediated tumor promotion (Scribner and Slaga, 1973). Fluocinolone acitonide is as effective as dexamethasone at lower dose levels and also blocks promoter-induced hyperplasia and DNA synthesis (Schwarz et al., 1977).As summarized above, glucocorticoids also inhibit expression of acylhydrolase activity and consequently prostaglandin production only in some cells (Hong and Levine, 1976; Tam et al., 1977) and tissues (Blackwell et al., 1980; Flower and Blackwell, 1979; Nijkamp et al., 1976). Indomethacin, a more potent inhibitor of prostaglandin production but a weak inhibitor of acylhydrolase activity (Ohuchi and Levine, 1978b), at low doses is only a weak inhibitor of TPA-induced tumor promotion (Viaje et al., 1977) but at high doses is an effective inhibitor of TPA-induced biochemical effects (Fiirstenberger and Marks, 1978; Verma and Boutwell, 1977). Since indomethacin can affect acylhydrolase activity, mediation of the TPA effects by the lipoxygenase products of arachidonic acid transformation (e.g., hydroxy fatty acids and leucotrienes) cannot be excluded. D. STIMULATIONOF PROSTAGLANDIN PRODUCTION BY GROWTH FACTORS Some proteins stimulate acylhydrolase activity and prostaglandin production, e.g., epidermal growth factor and platelet-derived growth factor (Coughlin et al., 1980; Levine and Hassid, 1977a). Several growth factors recently have been described and characterized (Dayer et al., 1977; DeLarco and Todaro, 1978; Meats et al., 1980; Roberts et al., 1980). All these factors stimulate prostaglandin production (Levine, 1981); they probably do so by stimulating acylhydrolase ac-

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

67

tivity. Some of these growth factors have been shown to confer transformed phenotypes on normal cells in vitro (DeLarco and Todaro, 1978; Roberts et al., 1980). A family of such growth factors probably exists in cells; their mitogenic effects and ability to transform phenotypes may be causally related to their capacity to stimulate acylhydrolase activities. VI. Prostaglandins: Their Effects on Cell or Tumor Growth

Addition of exogenous prostaglandins to cells in culture has led to the conclusion that prostaglandins, especially PGE, inhibit cell growth. PGE, added to cultures of mouse 3T3 fibroblasts slows growth of the cells (Johnson and Pastin, 1971, 1972). The growth of murine plasma tumor (MPC-11) is suppressed by 0.1 to 10 pg PGE,; PGE2 is slightly less effective; and PGF2, is ineffective (Naseem and Hollander, 1973). PGE inhibits the growth of mouse EL-4 lymphoma cells; PGEZ, 1-100 pg/ml, also inhibits proliferation as measured by thymidine uptake (Sonis et al., 1977). PGEl, 1 puglml, inhibits cell replication by mouse B-16 melanoma, and in vivo 16,16-dimethylPGEz methylester, 5 pg injected subcutaneously at the site of the tumor cell injection, reduces tumor growth (Santoro et al., 1976). PGF2a, 10-400 ng/ml, added to quiescent Swiss mouse 3T3 cell initiates DNA synthesis and cell proliferation in a small proportion of the cells. PGEl and PGE2also are effective at initiating DNA synthesis but only at high concentrations, and even at high concentrations these prostaglandins are less effective than PGFza (DeAsua et al., 1975). Inhibitors of prostaglandin production also have been used to study the effects of prostaglandins on tumor growth. In general, they stimulate growth, but inhibition also has been observed. It must be recalled that inhibitors of cyclooxygenase by nonsteroidal anti-inflammatory drugs block the production of prostaglandins, prostacyclin, and thromboxanes. Inhibitors such as 5,8,11,14-eicosatetraynoicacid block production of cyclooxygenase and lipoxygenase products. Antiinflammatory steroids also may block the production of cyclooxygenase and lipoxygenase products. Some inhibitors (e.g., indomethacin) have multiple specificities and may affect enzymes other than those mediating prostaglandin synthesis. Indomethacin (0.1-100 nM) increases the replication of HEp-2, L, and HeLa cells; the effect is reversed by adding PGEl (1 pg/ml) to the culture. However, indomethacin at 1p M does not significantly alter growth (Thomas et al., 1974).The growth of mouse B-16 melanomain vitro is enhanced in the presence of indomethacin or hydrocortisone (Santoro et al., 1976).Rep-

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lication of methylcholanthrene-induced fibrosarcoma cells from C3H mice is not significantly affected by 0.1 pgfml indomethacin, but at 20 kg/ml, thymidine uptake is increased 2.5-fold (Lynch et aZ., 1978). On the other hand, indomethacin, aspirin, or sodium salicylate reversibly inhibit growth of rat hepatoma cells; there is no effect with 1-10 p M indomethacin, approximately 20% inhibition with 0.1 mM, and 80% inhibition with 0.5 mM (Hial et al., 1977). Indomethacin does not affect the growth of mouse HSDM, fibroblasts in vitro (Levine et al., 1972), but indomethacin (100-125 pg) administered to mice daily with their food reduces the weights of the HSDMl fibrosarcoma by 27% (Tashjian et al., 1973).Indomethacin (50 pg administered subcutaneously on alternate days) reduces the size of Maloney sarcoma virus-induced tumors in 20-day-old BALB/c mice and delays the onset of tumor growth (Humes et aZ., 1974). In older mice (6-week-old), the same dose of indomethacin is much more effective at reducing the size of the Maloney sarcoma virus-induced tumors (Strausser and Humes, 1975). Bone destruction by the virus-induced tumor is also inhibited by this indomethacin treatment. The growth of a transplanted methylcholanthrene-induced mouse fibrosarcoma is reduced in mice given indomethacin (125 pg/day) interperitoneally for the first 10 to 14 days (Plescia et al., 1975). Treatment of mice with aspirin is also effective at reducing the growth of tumors; aspirin (150 mg/kg administered twice daily by mouth) inhibits the growth of the transplanted mast-cell ascites tumor (P185)and Lewis lung carcinoma. Indomethacin (3-5 mg/kg) is even more effective (Hialet aZ., 1976). In C3H mice bearing a transplantable methylcholanthrene-induced fibrosarcoma, indomethacin or aspirin (administered by way of drinking water from day 7 to day 49 after tumor transplantation) reduces the tumor size measured at day 46 (Lynch et al., 1978). Indomethacin, aspirin, or hydrocortisone also increase survival time of the mice. The nonsteroidal anti-inflammatory drug flurbiprofen, an effective inhibitor of prostaglandin production, in combination with chemotherapy or radiotherapy reduces tumor growth in mice when compared to the treatment by chemotherapy and/or radiotherapy alone (Bennett et al., 1979). In addition, flurbiprofen given with methotrexate in mice following removal of the primary tumor prolongs survival and reduces the incidence of local recurrence (Berstock et al., 1980).Indomethacin inhibits growth of fibrosarcoma in mice and potentiates immunotherapy (Lynch and Salomon, 1979). Administration of indomethacin to rats with Yoshida hepatoma cells reduces tumors (Trevisani et al., 1980). N-(4-Hydroxyphenyl)retinamide, an effective inhibitor of prostaglandin production (Levine, 1979a), inhibits the development

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

69

of breast cancer induced in rats by N-nitroso-N-methylurea when fed orally to rats over a 2-week or 6-month period (Moon et al., 1979). Prostaglandins may mediate metastatic spread of tumors, especially to bone. A correlation between the level of PGE2production by human breast cancer and its metastasis to bone has been reported (Bennett et al., 1975). In rats bearing a Walker carcinosarcoma, treatment with aspirin or indomethacin prevents metastasis to bone but does not slow growth of the tumor (Powles et al., 1978). In rabbits, indomethacin slows the skeletal destruction from tumor metastases but does not affect the development of pulmonary metastases (Galasko and Bennett, 1976). Development of osteolytic bone tumors in rats can be prevented by aspirin and indomethacin (Dowsett et al., 1976a,b; Powles et al., 1973). Benoral, an aspirin-paracetamol conjugate, also prevents bone metastasis in rats (Powles et al., 1980). However, treatment of human patients with breast cancer with aspirin and indomethacin does not significantly reduce metastasis to the bone (Powles et al., 1980). PGD, may play a role in metastases. This conclusion is based on the findings that the highly metastatic malignant melanoma B16Flo forms less PGD2 from PGH, compared to the moderately metastatic parent cell line B 16F1(Fitzpatrick and Stringfellow, 1979). VII. Prostaglandins and the Immune Response

As documented in earlier sections of this review, addition of exogenous prostaglandins or inhibitors of prostaglandin synthesis to cells in culture or in vivo affects cell and tumor growth. The use of inhibitors with multiple specificities, the number of arachidonic acid metabolites (Fig. l), their diverse pharmacological activities, and their lability may explain some of the contradictory findings attributed to a particular metabolic product or inhibitor. Nevertheless, it is clear that neoplastic tissues produce high levels of prostaglandins, probably more than normal tissue. It is also apparent that prostaglandins or inhibitors of prostaglandin synthesis affect cell and tumor growth. In Section VI, several examples of slowing tumor growth in vivo by the administration of inhibitors of prostaglandin synthesis have been cited. One general mechanism that could account for many of these phenomena is that prostaglandins affect the immune response; i.e., increased prostaglandin production suppresses the host’s immune response to the tumor. Inhibition of prostaglandin production by anti-inflammatory drugs (e.g., indomethacin, aspirin, flurbiprofen, flufenamic acid, glucocorticoids, or 5,8,11,14-eicosatetraynoicacid) permits the host’s

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immune system to more effectively reject the tumor. Such a view is supported by the following findings. Mice bearing a virus-induced ascites cell tumor (MCDV-12) or a methylcholanthrene-induced fibrosarcoma (MC-16) do not produce antibodies to sheep erythrocytes. The tumor cells block the immune response to the sheep erythrocytes when added to spleen cells in the presence of the sheep erythrocytes. These tumors synthesize large amounts of PGE2, and PGEz also blocks production of antibodies to the sheep erythrocytes in this in vitro system (Plescia et al., 1975). Administration of indomethacin, flufenamic acid, and aspirin to the syngeneic C57B1/6J mouse blocks the immunosuppressive activity of MC16 cells in vivo (Grinwich and Plescia, 1976). In untreated mice bearing MeC3-1 tumors, no evidence of immunosuppression is found; an enhanced immune response to sheep erythrocytes is reported (Lynch et al., 1978). In tumor-bearing mice, treatment with indomethacin phrtially restores the depressed mitogen responses of spleen cells to phytohemagglutinin and to bacterial lipopolysaccharides (Pelus and Strausser, 1976). Inhibitors of prostaglandin synthesis enhance the natural and antibody-dependent cytotoxicity of lymphocytes for PGEz-producing tumor cells (Droller et al., 1978). PGEz induces suppressor T lymphocytes (Goodwin and Webb, 1980) and also the production of suppressor factor by lymphocytes (Rogers et d., 1980). The source of the PGEz may not be the tumor cells. Macrophages secrete PGEz (Kurland and Bockman, 1978) which may regulate the proliferative response of normal peripheral blood mononuclear cells to T-cell mitogens (Goodwin et al., 1977, 1978). In the presence of indomethacin, the mitogen-induced lymphocyte proliferation is increased (Goodwin and Messner, 1979). PGEz overcomes this enhancement and returns the proliferative response to normal. In mice injected with sheep erythrocytes, administration of indomethacin increases the appearance of anti-sheep erythrocyteproducing spleen cells (Webb and Osheroff, 1976); i.e., the inhibitors of prostaglandin synthesis act on the suppressor cells. Lymphocytes from patients with Hodgkin’s disease incorporate less thymidine and produce increased levels of PGEz in response to phytohemagglutinin (Goodwin et al., 1977). Inhibitors of prostaglandin synthesis block both of these effects. In ten anergic patients with a variety of metastatic solid tumors, indomethacin does not reverse the depressed phytohemagglutinin response in vitro (Kauffman et al., 1978). However, an increase in prostaglandin-mediated immunosuppression may be an important factor in the anergy of some patients with malignancies other than Hodgkin’s disease (Vosixa and Thies, 1979).

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VIII. Challenges

Most cells have the potential to deacylate cellular lipids, to convert the free arachidonic acid to endoperoxides, and to form lipoxygenase-mediated hydroperoxy fatty acids. The endoperoxides are enzymatically and nonenzymatically converted to prostacyclin, thromboxane, and PGE,, PGF,, and PGD2, whereas the hydroperoxyarachidonic acid can be converted to several hydroxy fatty acids, dihydroxy fatty acids, and/or to a family of fatty acids that contain glutathione, cysteinylglycine, or cysteinyl moieties [ slow reactive substances (SRS)]. Many of these end products are unstable chemically and/or metabolically. Most of them are pharmacologically potent and possess diverse activities. This biochemical cascade is controlled at the deacylation reaction since free arachidonic acid is not present in cells; it is esterified. The deacylation reactions that make free arachidonic acid available for the cyclooxygenases and lipoxygenases are triggered by a variety of compounds of diverse biological activities (Levine, 1981). Protein synthesis is required for many of these stimulations; the rapidly turning over protein could be the phospholipase or a protein regulator(s) of phospholipase activity (Pong et al., 1977). We also suggest as one mechanism of action of anti-inflammatory steroids that “the steroids could affect the synthesis of a regulator for the phospholipase activity” (Tam et al., 1977). Such a regulator has been partially purified (Blackwell et d,1980; Hirata and Axelrod, 1980). Most likely, since there is more than one reaction leading to deacylation, more than one regulator of deacylation will be found. Most cells also have the enzymatic capacity to transform the free arachidonic acid. The final products of this transformation are unique to the cell; i.e., the levels and proportions of each product synthesized by the cell are intrinsic properties of that cell (see Table I). A cell that produces high concentrations and proportions of PGIz will produce predominantly PGI, when stimulated.. A cell that produces predominantly PGEz or TxAz will produce PGEz or TxAz in the same proportion when stimulated. It is not improbable that cells of similar embryonic origin but from different species will synthesize similar proportions of arachidonic acid transformation products. Cells also differ in their sensitivity to stimulation; for example, among the cells listed in Table 11, the TPA concentrations needed to stimulate vary by 2 to 3 orders of magnitude (L. Levine, unpublished data). Therefore, it is not surprising that some of the published effects of prostaglandins and prostaglandin synthesis inhibitors on cell and tumor growth conflict. Most of the studies cited suggest that

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arachidonic acid transformation is associated with cancer. The evidence for this association is (1)that neoplastic tissue produces high levels of prostaglandins, (2) that prostaglandins affect cell growth in vitro and tumor growth in vivo, and ( 3 ) that arachidonic acid transformation is associated with tumor promotion. However, whereas nonsteroidal anti-inflammatory inhibitors of prostaglandin synthesis may reduce tumor growth i n vivo or tumor growth in the two-stage mouse skin model of carcinogenesis, their effects are weak at best. Steroids are much more effective in the latter model. The effects of the nonsteroidal anti-inflammatory inhibitors of prostaglandin synthesis on tumor growth, i n vivo, are best observed when combined with chemotherapy or radiotherapy. Nevertheless, a causal relationship between arachidonic acid transformation and cancer has yet to be demonstrated. At least three steps in the arachidonic acid transformation scheme can be considered as being responsible for the previously summarized associations:

1. Acylhydrolase activity can lead to production of lysophospholipids and alteration of membrane fluidity (Hirata and Axelrod, 1980) [TPA treatment, which stimulates deacylation of cellular lipids (Levine, 1981), also alters membrane fluidity (Castagna et al., 1979; Weinstein et al., 1979)l. 2. Cyclooxygenase activity leads to production of the prostaglandins, prostacyclins, and thromboxanes. 3. Lipoxygenase activity leads to formation of the hydroxyarachidonic acids and SRS compounds. As noted, inhibitors of cyclooxygenase activity are only partially effective, at best, at reducing tumors, whereas inhibitors of acylhydrolase activities such as glucocorticoids, which also block reactions (2) and (3), are more effective. Thus, acylhydrolase activity, or possibly lipoxygenase activity, is more likely to be causally related to cancer than cyclooxygenase activity. It is possible that all three activities combine to give the tumor cell a favorable advantage for growth; the altered fluidity increases growth potential and differentiation properties of the cell, and, at the same time, the cell is stimulated to synthesize products that inhibit the immune response. It should be recalled that neoplastic tissue does produce high levels of prostaglandins; thus, acylhydrolase activity of neoplastic tissue and alteration of membrane fluidity is correspondingly high. These considerations suggest that inhibitors of acylhydrolase activity would be effective at reducing tumor growth i n vivo. Although the glucocorticoids inhibit the expression of acylhydrolase activity, they

ARACHIDONIC ACID TRANSFORMATION AND TUMORS

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do so in some but not all cells; and, in addition, they may affect biochemical reactions other than expression of acylhydrolase activities. The regulators of acylhydrolase activity may be more specific. If arachidonic acid transformation and cancer are indeed causally related, an understanding of how these regulating molecules exert their effects may lead to their use in the control of tumor growth in uiuo. At any rate, the elucidation of their mechanism of action is required and offers a challenging area for future research.

ACKNOWLEDGMENTS The author’s research is supported by grants GM-27256 and CA-17309 from the National Institutes of Health. He is an American Cancer Society Research Professor of Biochemistry (Award PRP-21). Thanks are due to Professor Helen Van Vunakis for critically reading the manuscript.

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