Prostaglandins and Host Defense in Cancer

Symposium on Prostaglandins

Prostaglandi ns and Host Defense in Cancer James S. Goodwin, M.D. *

The aim of this article is to review the data linking prostaglandin production to the growth of cancer in humans and experimental animals. Because this area of research is rife with conflicting data, it may be valuable for the reader if I state my biases at the outset. I feel that the preponderance of data suggests that production of prostaglandins by tumors or by host immunocytes is one means by which tumors can escape immunologic rejection. In other words, prostaglandin production, particularly PGE production, is good for the tumor and bad for the host. I should add that not all experiments, nor all investigators, support this concept; so I will attempt to present both the supporting and conflicting data and interpretations. The concept that prostaglandins act ·predominately to promote tumor growth has an obvious corollary of potentially great importance: that prostaglandin synthetase inhibitors might serve as anti-tumor agents. The concept that prostaglandin synthetase inhibitors (PGSI) might act as immunoadjuvants in the treatment of cancer rests on the belief that endogenous prostaglandins in general act as immunodepressants. The role of prostaglandins in immunoregulation will be briefly reviewed in this paper and has been discussed in more detail elsewhere. 1 If we accept the concept that endogenous prostaglandins are indeed immunodepressants, then we dm derive five arguments why prostaglandin synthetase inhibitors might be useful in cancer patients. 1. Prostaglandin synthetase inhibitors in vitro and in vivo act as general immunostimulants. 2. Tumors induce increased prostaglandin production by host macrophages, leading to immunosuppression. 3. Prostaglandin production by tumor cells can cause a direct, local suppression of host immunocytes. 4. Immunoadjuvants such as C. parvum and BCG cause induction of prostaglandin production in the host, leading to an inhibition of the immunoadjuvant effects. 5. Prostaglandins may play a role in the establishment of tumor metastases.

I will discuss the evidence supporting each of these arguments in turn and then focus on the data which conflict with these ideas. "Associate Professor of Medicine and Chief, Division of Gerontology, University of New Mexico School of Medicine, Albuquerque, New Mexico

Medical Clinics of North America - Vo!. 65, No. 4, July 1981

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PG Synthetase Inhibitors Act as General Immunostimulants The early attempts to define physiologic roles for prostaglandins were frustrated by the fact that prostaglandins are ubiquitous. It was difficult to assign specific functions to compounds that were produced by every tissue in the body. More recently it has been appreciated that prostaglandins are purely local hormones. The prostaglandins produced in a given tissue act on that tissue to modulate its function. Moreover, in the physiologic systems where the role for prostaglandins has been clearly defined, these compounds, particularly PGE, act as local feedback inhibitors.2 For example, vasopressin stimulates the production of PGE 2 in the distal nephron; this PGE 2 in turn makes the collecting duct less sensitive to the action of vasopressin in increasing water permeability.3 Analogous roles for PGE 2 have been described in gastric acid secretion,4 adrenergic synaptic transmission,s and release of insulin from the pancreas. 6 Blockade of prostaglandin production by the administration of indomethacin or other PG synthetase inhibitors causes increased concentrating ability of the kidney, increased gastric acid output, increased norepinephrine release from adrenergic neurons, and increased insulin release. 3.4 In a way, prostaglandin synthetase inhibitors act as acrossthe-board physiologic stimulants, eliminating the PGE mediated feedback suppressor loop found in many tissues. Knowledge of a prostaglandin-mediated feedback suppressor system in humoral and cellular immune responses evolved in a manner that was similar to the investigations of PG actions in other tissues. In 1971 Smith et al. reported that PGE I and E 2 • as well as Al and Fin, inhibited 3H-thymidine incorporation into PHA-stimulated human lymphocytes. 7 Other investigators then showed in various in vitro assay systems that PGE I and E2 could depress macrophage inhibitory factor activity,S leukocyte inhibitory factor production,9 direct cytolysis by activated lymphocytes,lO hemolytic plaque formation,l1 and antibody formation. 12 One problem with these early studies was the high concentrations of prostaglandins used (10- 6 to 1O-4M) to demonstrate inhibition in vitro. Since the concentration of PGE in inflammatory sites is usually less than 1O-sM,13 it was difficult to know whether endogenous prostaglandins actually did inhibit immune function in vivo. For example, Berenbaum et alY concluded from their studies that the in~itory effects of prostaglandins seen in vitro represented a nonspecific toxicity from the high, unphysiologic concentrations of the agents used. 14 These doubts were finally dispelled by two sets of experiments performed in different laboratories, both showing a role for endogenous prostaglandins in immune regulation in vivo. Webb and Osheroff showed that intravenous injections of sheep red blood cells (sRBC) in mice resulted in an 80-fold increase in splenic PGF 2n within minutes of the injection. 15 Prior administration of indomethacin or other PG synthetase inhibitors to the mice prevented the PGF 2n increase and also resulted in a significantly increased number of direct plaque forming cells in the spleen 3 to 5 days after the sRBC injection. Thus prostaglandins exerted a negative regulatory role on formation of plaque forming cells in vivo after antigenic challenge. In addition Plescia and his co-workers found that the immunosuppression caused by certain syngeneic h!.mors in mice could be reversed in vitro by PG synthetase inhibitors.16 A more dramatic finding was that tumor growth could be slowed in vivo by administration of indomethacin to the mice.

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The experiments in mice by Webb and by Plescia provided strong evidence for a role for endogenous prostaglandins in immunoregulation in certain systems. These results prompted several groups of investigators to reexamine the question of what part prostaglandins might play in the control of immune responses in experimental animals and in humans. The data reviewed here will in general include only those studies where a physiologic, as opposed to a pharmacologic, effect of prostaglandin has been documented. The current understanding of immunoregulation by endogenous prostaglandins results from work by many laboratories using in vitro and in vivo systems in mice and man. PGE is produced by human peripheral blood mononuclear cells (PBMC) and by mouse splenocytes in response to stimulation' by mitogens or antigens, and the PGE 2 inhibits the subsequent T cell proliferative response. 17-26 Blockade of prostaglandin production in vitro results in enhanced T cell proliferation. 17-2o In human peripheral blood, the cell responsible for PGE production is probably of monoctye-macrophage lineage,19-22 while there is good evidence in mice that glass-adherent T cells produce large amounts of PGE.17. IS In some systems it would appear that a signal from activated T cells stimulates PGE 2 production by adherent cells. 27 Thus the feedback loop comes full circle: an anti genic stimulus induces proliferation and lymphokine production in sensitized T cells; some of these lymphokines induce PGE production by adherent cells (either macrophages or T cells) and this PGE inhibits further T cell proliferation and lymphokine production. . The mechanism of inhibition by PGE is not completely understood. Specifically, it is not clear whether PGE directly inhibits the responding cell or whether PGE acts via an intermediate cell; that is, does PGE activate a suppressor cell population which in turn inhibits the responding cells via a non PG-related pathway? Webb and associates have suggested that PGE produced by adherent mouse splenocytes causes nonadherent splenocytes to release suppressor factors which inhibit T cells proliferation. IS We have shown that only a small percentage of hu~an peripheral T cells have receptorsfor PGE, suggesting that inhibition by PGE might be mediated by activation of this subpopulation. 2s Both cellular and humoral immune responses are under negative control by prostaglandins. In vitro PGE inhibits T cell proliferation/9. 29lymphokine production,30 and the generation of cytotoxic cells. 31 In addition PGE causes T cells to become more adherent3 2 and to express Fc receptors. 33 In vivo, PG synthetase inhibitors result in enhanced delayed hypersensibility skin test responses 34 • 35 and in enhanced cytotoxicity.36 The control of humoral immune responses by prostaglandins is not as straightforward as the control of cellular immunity. While the addition of PG synthetase inhibitors in vitro23 . 37 and in vivo l5 . 38 results in increased plaque forming cells and/or antibody titer after immunization, the direct addition of prostaglandins to in vitro systems of immunoglobulin production or induction of plaque forming cells is generally not suppressive unless pharmacologic amounts of prostaglandins are employed. 39 Thus in humoral immunity, prostaglandins are necessary but not sufficient for inhibition; some other signal is also required. For the purposes of this review, however, the important fact is that administration of prostaglandin synthetase inhibitors to normal mice and man results in enhanced humoral immune responses.

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Some macrophage functions are also regulated by prostaglandins. Small concentrations of PGE (l0-9M) cause substantial suppression of macrophage spreading and adherence, while larger concentrations (l0-7 to lO- SM) enhanced migration. 40 PGE also inhibits macrophage tumoricidal activity, but the concentration of PGE necessary for inhibition (1O- 6M) makes it questionable whether this effect has physiologic relevanceY More recently unpublished work from one laboratory suggests that macrophage cytotoxicity is indeed under negative control by endogenous prostaglandins. PGE produced by monocytes can also act as a modulator of further monocyte differentiation. Picker et al.4 2 showed that monocyte-produced PGE regulates the change with time in culture of the specific activity of two cytoplasmic enzymes, 5 ' -nucleotidase and acid phosphatase. 42 Natural killer (NK) activity is under negative control by PGE 2. Roder and Klein showed that PGE inhibited and indomethacin enhanced murine NK activity against YAC target cells. 43 The PGE mediated inhibition was at the level of the NK cell and not the target cell. Brunda et al. 36 confirmed these results and extended them to show that PGE inhibited and indomethacin enhanced interferon-augmented NK activity.

Tumors Induce Increased PG Production in Host Macrophages, Leading to Immunosuppression In the previous section I briefly reviewed the evidence that PGs were feedback inhibitors of normal humoral and cellular immune responses. In this section I will show that this normal feedback system is accelerated in some tumor-bearing animals and man, leading to suppressed immunity. Pelus and Strausser first reported that the depressed mitogen response of splenocytes from tumor-bearing mice could be increased with the addition of indomethacin to the mitogen cultures. 44 My colleagues and I investigated whether overactivity of the normal prostaglandin regulatory system was responsible for the depressed mitogen response seen in patients with different cancers. Our first investigation concerned patients with Hodgkin's disease. Previous investigations by Twomey et al. 45 and Sibbitt et al.4 6 had suggested that the defect in cell-mediated immunity in Hodgkin's disease was caused by circulating suppressor cells. In 6 subjects with untreated Hodgkin's disease the mean PHA response was 48 per cent of the mean response of 28 controls.47 The addition of indomethacin to the PHA cultures caused a 44 ± 18 per cent increase in 3H-thymidine incorporation for the controls versus a 182 ± 60 per cent increase for the patients with Hodgkin's disease (mean ± SD, P < 0.001) (Fig. 1). When RO-20-5720, a PG synthetase inhibitor structurally unrelated to indomethacin48 was used, results were similar, with a 95 ± 29 per cent increase in the PHA stimulation of Hodgkin's disease lymphocytes versus a 28 ± 10 per cent increase for controls. After addition of indomethacin, the mean response of the Hodgkin's disease lymphocytes increased to 94 per cent of the mean control response. The suppression of the PHA response in the patients with Hodgkin's disease was also reversed by removal of glassadherent cells prior to culture. This was expected, since the PG-producing suppressor cells are glass-adherent. 19 Two possible explanations for the increased response of Hodgkin's disease lymphocytes to indomethacin are apparent. First, the Hodgkin's

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20,000 15,000 Figure 1. Phytohemagglutinin (PHA) response (mean ± S.E.M.) in counts per minute (cpm) of 6 patients with Hodgkin's disease and 20 normal controls with or without the addition of indomethacin. Without indomethacin, the patient and control group are significantly different (t = 3.01, p < 0.002). After indomethacin there is no significant difference (t = 0.33, p > 0.6 by onetailed test). Cpm are graphed on a log scale. (Reprinted with permission from the New England Journal of Medicine.)

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disease lymphocytes could be more sensitive to the prostaglandins produced by the suppressor cells. Second, th~ suppressor cells could be producing more prostaglandins. To investigate the first possibility, PGE 2 was added to cultures of PBMC from patients with Hodgkin's disease versus controls. No difference in sensitivity to inhibition by exogenous PGE 2between Hodgkin's disease and normal lymphocytes was found. PGE 2 production was then measured in mitogen cultures of PBMC from three patients with Hodgkin's disease and three age- and sex-matched controls. The mean PGE 2 production of the Hodgkin's disease PBMC was 20,600 ± 7,620 pg/ml (6 x lO-BM) in 48 hours versus 5,368 ± 2,944 pg/ml (1.6 x lO-BM) for the controls (mean ± SD, P < 0.02). Bockman has recently confirmed that adherent mononuclear cells from patients with Hodgkin's disease produce four times as much PGE 2 as normal. 49 He also showed that addition of indomethacin increases the number ofT-lymphocyte colonies formed on soft agar after PHA stimulation ofPBMC from patients with Hodgkin's disease. De Shazo has reported findings similar to ours, and has also shown that in vivo indomethacin treatment of patients wtih Hodgkin's disease increased the in vitro lymphocyte blastogenic responses but did not change the responses to delayed hypersensitivity skin testing in the four patients tested. 50 Thus in Hodgkin's disease overactivity of the normal PGE feedback inhibition system would appear to be a major cause of the T cell dysfunction. In other human cancers the evidence is less clear. In mice it would appear that several types of experimental tumors all induce increased PGE production by host macrophages,51 and this might also be the case in man. 52 We studied lO anergic patients with a variety of metastatic solid tumors and found that the enhancement of the mitogen responses with indomethacin was not greater than control. 53 Vosixa and Thies studied 24 patients with different cancers.54 Of the 6 patients with depressed mitogen responses, four had a substantial increase in response with in vitro indomethacin. However, even after indomethacin the mitogen responses were still depressed compared to

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control. Balch and Tilden tested 24 patients with melanoma and showed a larger increase in the patients' mitogen responses with in vitro indomethacin than in the controls. 55 Once again the patients' responses were still depressed after indomethacin. The important question of whether in vivo indomethacin administration would restore depressed cellular immune responses in cancer patients has not been systematically addressed. We have previously shown that in vivo indomethacin would restore skin test reactivity in two anergic patients with adult combined immunodeficiency (Fig. 2), but we have not performed similar studies in patients with cancer. 35 De Shazo found no effect of in vivo indomethacin on skin test reactivity in his four patients with Hodgkin's disease. 50

PG Production by Tumor Cells Can Cause a Direct Local Suppression of Host Immunocytes Several different laboratories have measured levels of prostaglandins in malignant tissues of humans and experimental animals. The initial experiments were performed by investigators interested in the mechanism of hypercalcemia associated with cancer not metastatic to bone. In 1970 Klein and Raisz showed that PGE 2 was as potent a stimulator of bone resorption in tissue culture as was parathyroid hormone. 56 Tashjian and his coworkers then showed that the hypercalcemia associated with certain nonmetastatic cancers in mice was caused by the tumor secreting large amounts of PGE 2 into the circulation. 57 Subsequent studies have linked overproduction of PGE 2 to some tumor-associated hypercalcemia states in humans (reviewed in 58). One result of this work was the demonstration that many naturally occurring or experimentally produced cancers synthesize large quantities of prostaglandins. High levels of PGE or PGE metabolites are found in the serum of patients with squamous-cell carcinoma of the lung,59 breast cancer,60 renal-

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Figure 2. Effect of indomethacin administration in vivo on the response to PHA in vitro and on the response to skin testing with four common antigens (SKSD, candida, trychophyton, mumps) in a patient with adult-acquired immunodeficiency. The two shaded areas represent the period of indomethacin administration (25 mg 4 times daily). Before indomethacin, the subject was completely unresponsive (no induration) on multiple occasions to the four antigens. With indomethacin administration the subject responded to two of the four antigens with > 10 mm induration. The in vitro response of the patient's peripheral blood mononuclear cells to an optimal concentration of PHA and to PHA plus indomethacin (1 JLglml) and also graphed. (Reprinted with permission from the Journal of Clinical and Laboratory Immunology).

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cell carcinoma,62 and a variety of other malignancies in man 59 ,62 and experimental animals,63, 64 In addition, some cancer tissues removed at operation contain more PGE 2 than normal tissue65 and synthesize large amounts of PGE 2 in vitro. 66·6B PGE 2 has been localized in human cancer tissue by means of immunofluorescence with rabbit anti-PGE antisera. 69 Strong immunofluorescence against PGE was detected in tumor cells in 27 of 42 malignancies. In addition, immunofluorescence against cyclic AMP, the putative second messenger for PGE,7° was found in 30 of the 42 malignancies. Because many different types of cancers produce large amounts of prostaglandins,71 it is logical to assume that these prostaglandins aid in the escape of these tumors from immune surveillance. The first and most exciting work in this regard was done by Plescia and his co-workers at Rutgers University.I6 These workers studied immune responses in mice bearing two syngeneic tumors - a virus-induced ascites cell line (MCDV-12) and a methycholanthrene-induced fibrosarcoma line (MC-16). Mice bearing these tumors were immunologically unresponsive to sheep RBC. Addition of the tumor cells to in vitro cultures of splenocytes and sheep RBC also suppressed the immune response, as measured by formation of plaque-forming cells. These tumors secrete large amounts of PGE 2/2 therefore the possibility that the immunosuppression was mediated via prostaglandin was investigated. The tumor-induced immunosuppression in vitro could be mimicked by the addition of PGE 2 in submicromolar concentrations. In addition, the tumorinduced immunosuppression in vitro and in vivo could be partially blocked by indomethacin I6 and other PG synthetase inhibitors.72 Perhaps the most exciting discovery was that tumor growth in vivo could be slowed by giving the mice daily injections of indomethacin. The obvious inference of this last finding is that when tumor-mediated immunosuppression is blocked with indomethacin, host-immune defenses slow the tumor growth. Other workers have since shown that indomethacin in vitro or in vivo partially restores the depressed mitogen responses of splenocytes to phytohemagglutinin (PHA) and to bacterial lipopolysaccharide in tumor-bearing mice. 44 More recently, Droller et al. have reported that PG synthetase inhibitors enhance natural and antibody-depe~dent cytotoxicity by lymphocytes against tumor cell lines that produce PGE 2 .73 Several laboratories have confirmed the finding of Plescia et al. that administration of indomethacin and other PG synthetase inhibitors results in a slowing of tumor growth in mice. 74-78 Hial et al. administered aspirin or indomethacin to mice that had received a mast cell ascites tumor or Lewis lung carcinoma. 74 Both drugs in normal therapeutic doses caused a substantial slowing of the growth of both tumors. At higher doses of indomethacin (5 mg/kg/day) there was elimination of the tumors in 50 per cent of the cases, but there were also signs of gastric toxicity and weight loss in the mice. In these studies, as in the ones by Plescia, the PG synthetase inhibitors were started simultaneously with tumor inoculation. When there was a delay between tumor inoculation and drug administration, the anti-tumor effects were less pronounced. Lynch et al. reported that indomethacin and aspirin inhibited the growth of a methylcholanthreneinduced fibrosarcoma in mice. In these experiments the drugs were not started until seven days after tumor inoculation, when the tumor was already palpable. 75 ,76 In approximately 20 per cent of the indomethacin treated mice

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the tumor completely regressed and did not reappear after cessation of the drug. Strausser and her colleagues have reported that indomethacin will slow the growth of already established Moloney sarcoma virus-induced leg tumors in mice. 77 If the drug is started at the same time as virus injection, no tumors develop. As mentioned above, the assumption behind all these studies is that increased prostaglandin production by the tumor (and also by host macrophages) inhibits the immune response of the host, especially in the immediate environment of the tumor, allowing the tumor to es~ape immunologic rejection. The anti-tumor action of the PG synthetase inhibitors is presumed to be by interrupting this escape of immunologic rejection. There is quite a bit of evidence supporting this theory. Plescia et al. showed that administration of any of a variety of different PG synthetase inhibitors to mice bearing fibrosarcomas resulted in restoration of the plaque forming response 16 and antibody response 72 after immunization with sheep RBC. Droller and his co-workers have shown that PGE 2 produced by human bladder tumor cell lines inhibited both natural and antibody dependent cytotoxicity of the tumor cells by human lymphocytes. 73 Addition of a variety of PG synthetase inhibitors resulted in an increase in both types of cytotoxicity against the tumor cell targets. Brunda et al. showed splenocytes from mice with Moloney sarcoma virus-induced tumors had severely depressed NK activity, and that this NK activity could be restored to normal by administration of aspirin or indomethacin to the mice. 36 Lynch et aI., while confirming that the administration of PG synthetase inhibitors does indeed slow the growth of or eliminate many experimental tumors, dispute the claim that these drugs work via stimulation of the host's immune system. 75 They showed that mice that had completely eliminated their tumors with indomethacin treatment were not immune to rechallenge with the same tumor. In addition, augmentation of the immune response in tumor bearing mice by indomethacin did not correlate with the anti-tumor effects of the drug. I feel that these data are fairly weak arguments against the immunoadjuvant explanation of the anti tumor effects of PG synthetase inhibitors. Specifically, these data are entirely consistent with the possibility that PG synthetase inhibitors work via augmentation of NK activity and macrophage cytotoxicity, neither of which demonstrates immunologic memory. In any case, it should be clear that the anti-tumor effects of PG synthetase inhibitors are secondary to inhibition of PG production; that is the only action that all of these agents (indomethacin, aspirin, RO-20-5720, flurbiprofen, xylenol, etc.) have in common, yet all possess anti-tumor effects.

Immunostimulants Such as C. parvum and BCG Cause Induction of PG Production in the Host, Leading to an Inhibition of the Immunostimulation The initial enthusiasm for the use of nonspecific immunostimulants in human cancer has diminished somewhat in the light of the unimpressive results of a number of clinical trials. Even in the work with experimental tumors in animals, the success of agents such as BCG and c. parvum is highly variable, depending on the tumor burden, route of administration, and

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dosage. 79 In a search for the reasons behind the disappointing results of immunostimulants in cancer treatment, investigators have found that C. parvum and BCG, in addition to nonspecifically enhancing humoral and cellular immune responses,80 also induce the evolution of suppressor cells in the host animals. 81 -84 Thus the early stimulation of immune responses is replaced by a later suppression. For example, C. parvum infection in mice is followed by an early (3 days post injection) augmentation of NK activity.85 However, by two weeks after inoculation the NK activity drops to about 20 per cent of that of untreated mice. 86 This depression in NK activity is caused by a splenic suppressor cell stimulated by C. parvum. 86 With the appreciation that "immunostimulants" actually had both positive and negative effects on immune responses, the disappointing results of the chnical trials of these agents in cancer patients became more understandable. Recent work has focused on the nature of the BCG and C. parvum-induced suppressor cells, with the thought that immunostimulant treatments might be designed which avoided or negated the secondary suppressor cell generation. The BCG-induced splenic suppressor cell in mice is adherent, phagocytic, and lacks the Thy-1 antigen. 87 This macrophage-like cell inhibits NK cell activity, the generation of cytotoxic T cells, mitogen or alloantigeninduced T cell proliferation, and the plaque-forming response to antigens. 81 84,86, S7 One possible mechanism for such macrophage-like suppressor cells is via production of prostaglandins. There are now some preliminary data suggesting that prostaglandins may indeed be important in the function of these cells. First, both C. parvum and BCG induce macrophages to increase their PG production more than 50-fold. ss , S9 Second, indomethacin would appear to eliminate the BCG-induced suppression ofNK activity. BCG causes macrophages to produce factors that stimulate and inhibit NK activity, and it would appear that the inhibitory factors are prostaglandins. 90 Tracey and Adkinson have shown that aspirin or indomethacin in vitro or in vivo augment the BCG-induced increase in NK activity.90 It remains to be seen whether in vivo indomethacin will prevent the late development of splenic suppressor cells and the concomitant late decrease in NK gctivity, T cell cytotoxicity and humoral immune responses following BCG or C. parvum administration. This whole question was given more relevance by the finding that in vivo indomethacin substantially increased the anti-tumor activities of C. parvum and BCG. In one report the injection of 10 9 killed C. parvum organisms into fibrosarcoma tumors at 10 days and then again at 17 days after grafting resulted in elimination of the tumor by day 50. 75 However, if the C. parvum injections were given at days 17 and 24, when the tumor was substantially larger, there was no effect on subsequent tumor growth. The addition of indomethacin to this latter regimen resulted in elimination of the tumor in 50 per cent of the cases. Similar findings were reported for BCG. When the C. parvum was given by intraperitoneal injection rather than intratumor, no effect on tumor growth was noted unless indomethacin was also administered, in which case the tumors regressed in 50 per cent of the mice. PG synthetase inhibitors have also been combined with standard chemotherapeutic agents in cancer therapy. Powles et al. studied a chemoresistant variant of the Walker tumor. 91 Neither chlorambucil alone nor PG synthetase inhibitors (indomethacin or flurbiprofen) had a measurable effect on survival

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of mice given this tumor, while chlorambucil plus a PG synthetase inhibitor resulted in long term survival in 40 per cent of the animals. These same authors reported that indomethacin prevented bone marrow toxicity from another commonly used anti-cancer therapy, radiation, and also lessened the gastrointestinal toxicity of melphelan. 91 Another potential role for PG synthetase inhibitors is in combination with interferon. Interferon has been shown to have antitumor properties in vivo, and this has been related to its ability to enhance NK activity in vivo and in vitro. 92 ,93 It is also well recognized that interferon is a powerful stimulus for prostaglandin production in some tissues,94 and there is some evidence that prostaglandins may mediate the antiviral actions of interferon. 95 ,96 It is possible that interferon-stimulated PG synthesis negatively modulates its anti-tumor action, and that interferon plus a PG synthetase inhibitor would be a powerful combination. This is merely speculation and should be considered in the light of some evidence which conflicts with the whole concept of PG synthetase inhibitors as anti tumor agents, to be discussed later.

Prostaglandins May Have a Role in Tumor Metastases The last of the five reasons why PG synthetase inhibitors may prove useful in cancer therapy is that PGs may be important in the establishment of metastases. The evidence for this is largely circumstantial. Bennet et al. measured PG content of tissue from 66 breast carcinomas and 16 benign breast tumors. 66 ,67 Malignant tissue had higher PG content than the benign tumors, and the level of PG tended to be higher in those malignancies that were histologically classified as invasive. In addition, breast cancer tissue from women with bone metastases contained significantly higher levels of prostaglandin than breast cancer tissue from women without metastases, Rolland et al. 68 have reported similar results in a series of 91 women with breast cancer. These authors measured PGE production per mg protein in the microsomal fraction of homogenates of breast cancer tissue. They found that high PGE production was associated with tumor invasiveness as assessed by the presence of neoplastic cells in locallymphatics and axillary lymph node"s. This relationship remained statistically significant after controlling for tumor size. Tumors that were walled in by dense connective tissue had low PGE production. Two preliminary reports have shown that administration of aspirin inhibits metastatic spread of a number of experimental tumors in mice,97,98 but it is not clear from this work whether the effect of aspirin on metastases was due to inhibition of PG synthetase or came about via another mechanism - perhaps by acetylation of membranes. PGE is necessary for the production of osteoclast-activating factor by leukocytes,99 and PGE by itself is a powerful stimulus to bone resorption;100 therefore one could imagine that inhibition of PG synthesis might suppress the development of bone tumors. Strausser and Humes demonstrated a reversal with indomethacin of the hypercalcemia and also of tumor growth of a sarcoma in mice. 77 Powles et al. reported that aspirin or indomethacin would prevent osteolytic bone deposits, and hypercalcemia, but not soft tissue deposits of Walker tumor in rats. 101 In another study, indomethacin slowed the skeletal destruction from tumor metastases in rabbits, but the drug did not inhibit the development of pulmonary metastases, 102

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Evidence Against the Concept of PG Synthetase Inhibitors as Anti-Tumor Agents In the above discussion I have avoided mention of any conflicting evidence that would tend to negate the overall argument that PG synthetase inhibitors might be useful anti-tumor agents. Most of these conflicting experiments have utilized one experimental tumor caused by the B-16 melanoma cell line. While PGE inhibits the proliferation of normallymphocytes, it also slows the growth of many other cells including tumor cell lines, and the growth of these lines is stimulated by the addition of PG synthetase inhibitors.103 Santoro et al. reported that PGE inhibited and indomethacin stimulated the growth of B-16 melanoma cells in vitro. 104 In addition, the daily injection of a PGE analogue into the site of tumor cell inoculation in mice slowed the growth of the tumor by about 60 per cent. This same group of investigators has more recently reported that treatment of mice bearing B-16 melanomas with a PGE analogue causes partial restoration of the depressed delayed hypersensitivity and splenic plaque-forming responses, while treatment with indomethacin either had no effect or caused further depression of the immune response. 105 Administration of indomethacin also resulted in an accelerated development of palpable B-16 subcutaneous tumors. In another study using the B-16 melanoma cell line, Stringfellow and Fitzpatrick showed that PGD 2 production by these tumor cells was inversely correlated with their metastatic potential. 106 Exposure of the melanoma cells to indomethacin prior to inoculation resulted in a substantial increase in the rate of pulmonary metastasis. Jubiz et al. reported that daily injections of PGF 2" inhibited the growth of a hormone-dependent mammary tumor in mice. 107 This effect was associated with a reduction in serum progesterone concentrations and was abolished by concomitant progesterone injections, suggesting that the PGF 2" worked by inhibiting endogenous progesterone synthesis. It is clear that the reports reviewed in this section contradict the data presented earlier in this review. It is impossible at this stage to develop a comprehensive theory of the actions of prostaglandins and cancer, given the conflicting data. The B-16 melanoma line is unusual in that the major cyclo-oxygenase metabolite is PGD 2 rather than PGE.106 Certainly the number of experimental tumors whose growth is slowed by PG synthetase inhibitors in vivo is greater than the number whose growth is augmented. However this leaves open the very important question of which type of experimental tumor, if any, human cancers will most approximate. There are just not enough data on this point. A recent report would suggest however, that many human tumors react to PGs and PG synthetase inhibitors in a manner similar to most experimental tumors. Buick et al. studied the clonigenic capacity of tumor cells obtained from malignant effusions from 31 patients with epithelial tumors, predominantly ovarian and breast cancer. 1OB They found that host macrophages stimulated the growth of these cells in vitro and that PG synthetase inhibitors eliminated this stimulation. The experimental system employed was sufficiently unique to render any direct analogy to other systems impossible, but one possible inference from these data is that cyclo-oxygenase metabolites stimulated the growth of these cancer cells.

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CONCLUSION I would like to end this review by discussing the question of whether PG synthetase inhibitors should be employed in clinical trials in patients with cancer. To my knowledge no such controlled trials are presently underway, though a few anecdotal reports have appeared. Powles et al.' mentioned that they administered flurbiprofen plus standard chemotherapy to 3 patients with breast cancer unresponsive to chemotherapy alone. 91 They stated that the combination of chemotherapy plus PG synthetase inhibitor resulted in an "improved response" in all cases, but gave no details. Other single case reports have appeared as letters in various journals. One advantage of PG synthetase inhibitors is their relative safety and low toxicity l09 especially compared to cancer chemotherapeutic agents. There are 3 PG synthetase inhibitors among the 20 most commonly prescribed medications in the United States. The question of what type(s) of cancer should be the subject of such a trial is difficult. Based on the data from animal studies, the likeliest success would be found in cancers that produce large amounts of PGE, or in cancers that are being treated with BCG or C. parvum. Certainly a much more systematic study of PG production by all types of human cancers is needed, as well as a careful quantitation of the types of prostaglandins produced. Because of the work with B-16 melanoma tumors in mice, one would certainly be wary of giving indomethacin to patients with tu mol'S that produce large amounts of PGD 2 rather than PGE, if indeed such tumors occur in humans. It would of course be easier to pick certain human cancers for clinical trials with PG synthetase inhibitors if more data from experimental animal tumors were available. There is a pressing need for more experiments studying the possible synergistic effects of PG synthetase inhibitors combined with C. parvum, BCG, interferon, and the newer immunoaugmenting agents. In addition the intriguing results of Powles et al. showing not only a synergistic effect of PG synthetase inhibitors plus chlorambucil, but also. a decrease in side effects of cytotoxic therapy with PG synthetase inhibitors, need to be tested in many more experimental models. I feel that the whole relationship between prostaglandins and cancer has not received the attention it should from experimental oncologists. This may be because the whole area of prostaglandins is immensely confusing, with different cyclo-oxygenase metabolites having completely opposite results. Thus when one adds a cyclo-oxygenase inhibitor to a system, any result is possible. It is not even settled whether PGE has a physiologic role or is merely a metabolite of an unstable intermediate compound that is the "real" PG, or whether the effects of cyclo-oxygenase inhibitors are because of inhibition of production of the classic prostaglandins (PGE and PGF) or by inhibition of production of thromboxanes, prostacyclin, etc. This lack of precise definition of the system tends to discourage precision in experimentation, which in turn tends to keep careful investigators out of the area. The investigator studying the effects of PG synthetase inhibitors on cancer growth must accept these limitations.

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