Prostratili, a Nonpromoting Phorbol Ester, Inhibits Induction by Phorbol

(CANCER RESEARCH 51, 5355-5360, October 1, 1991] Prostratili, a Nonpromoting Phorbol Ester, Inhibits Induction by Phorbol 12-Myristate 13-Acetate of Ornithine Decarboxylase, Edema, and Hyperplasia in CD-I Mouse Skin Zoltan Szallasi and Peter M. Blumberg1

Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892

ABSTRACT (11, 12) and Marks (13) showed that tumor promotion could be subdivided into distinct stages differing in structure-activity Pretreatment of CD-I mouse skin with prostratin (12-deoxyphorbol requirements; mezerein and 12-0-retinoylphorbol 13-acetate, 13-acetate) inhibited biological response to phorbol 12-myristate 13- although only weakly promoting themselves, were effective if acetate. The three responses examined were hyperplasia, induction of preceded by one or more applications of PMA. ornithine decarboxylase, and edema; the characteristics of inhibition depended on the specific response. Hyperplasia is the best short-term In all of the above cases, the compounds induce in cultured correlate of tumor promotion. Two or more pretreatments with 2.56 jtmol cells essentially the complete spectrum of phorbol ester re (1 mg) prostratin, administered at intervals of 1-4 days, almost com sponses. This is not so for the bryostatins, a structurally distinct pletely blocked the hyperplasia induced by phorbol 12-myristate 13- class of protein kinase C activators. The bryostatins induce only acetate applied IS min to 6 h after the last pretreatment. Induri hilily of some of the responses seen for the phorbol esters; when coap- hyperplasia was partially restored at 2 days and recovered by 4 days. plied with the phorbol esters, the bryostatins block those re Prostratin was more potent for inhibition of ornithine decarboxylase sponses which they themselves do not induce (6). In mouse induction (50% inhibitory dose = 25.6 nmol) than it was for hyperplasia: keratinocytes, the bryostatins fail to induce markers of differ the inhibition was largely attained by the first application, and the entiation (14). In mouse skin, the bryostatins are inactive as recovery from inhibition was slower (8 days). Edema was partially inhibited by prostratin (dose giving 50% of maximal inhibition = 512 first stage promoters (15), very weak as second stage promoters nmol). We have previously demonstrated that prostratin is a protein or as complete promoters (16), strong inhibitors of first stage kinase C activator. Our present results show that prostratin is a functional promotion (15), and modest inhibitors of complete promotion antagonist for a class of protein kinase C mediated responses. The (16). findings emphasize the diversity of biological outcome for protein kinase One other report of protein kinase C activators which inhibit C activators, presumably driven by the extensive heterogeneity in the tumor promotion remains difficult to evaluate. Schmidt and protein kinase C pathway. Hecker (17) reported that coapplication of PMA and a 4-8- fold higher dose of phorbol 12,13-diacetate, -dibutyrate, -dipro- INTRODUCTION pionate, or -dibenzoate completely blocked promotion in NMRI mice. This was clearly not seen for phorbol 12,13- In mouse skin, phorbol esters exert tumor promoting, inflam diacetate in SENCAR mice (11), and phorbol 12,13-dibutyrate matory, and hyperplastic activity. These responses are thought and phorbol 12,13-dibenzoate are themselves tumor promoting to be mediated via the major phorbol ester receptor, PK.C2 (1- (18, 19). In the same paper, Schmidt and Hecker (17) also 4). Using [3H]PDBu, Blumberg and coworkers (5) characterized reported that a single cotreatment had no effect on thymidine specific, high affinity phorbol ester binding sites in particulate incorporation or hyperplasia in NMRI mouse skin and that, preparations from mouse skin and mouse epidermis. The meas after eight cotreatments, there was only a slightly less intense urements yielded curved Scatchard plots, consistent with recep hyperplasia than after eight treatments with PMA alone. tor heterogeneity. These were the first results suggesting that We have recently begun the detailed characterization of the the phorbol ester receptors, subsequently identified as PKC, biological effects of 12-deoxyphorbol 13-acetate (prostratin), might represent a family of isoforms differing in structure- motivated by its identification in a screen of natural products activity relations. It is now known that protein kinase C indeed with anti-human immunodeficiency virus activity.3 Prostratin consists of at least nine isozymes (6). The binding characteris was initially identified as a toxic constituent of Pimelea pros tics of only the a, /3, and 7 isozymes have been investigated in trata, an edemic New Zealand shrub implicated in the poisoning detail so far. These three isozymes appear quite similar for of livestock in New Zealand (20). recognizing the phorbol esters, although differences in interac Hecker and coworkers (21) reported that prostratin was tion with unsaturated fatty acids have been noted (7, 8). approximately 144-fold less potent than PMA for inducing Despite the lack of biochemical understanding, whole animal mouse ear reddening, and, at a dose 40-fold higher than that of analysis argues strongly for heterogeneity in response to the PMA, failed to be tumor promoting. Other doses and dose phorbol esters. A decade ago, Hecker and coworkers (9) dem schedules were not reported. In our initial analyses,3 we sought onstrated that 12-deoxyphorbol 13-phenylacetate, -isobutyrate, to characterize the spectrum of action and potency of prostratin or -angelate was inflammatory but either not promoting or in more detail; of particular importance for interpreting the weakly promoting. Similar behavior was noted for phorbol negative promotion experiments of Hecker was the adequacy esters with unsaturated side chains (10). The groups of Slaga of the dose used. We found that prostratin in vitro bound to protein kinase C with a K, of 12.5 HM (21-fold that of PDBu) Received 1/7/91; accepted 7/19/91. The costs of publication of this article were defrayed in part by the payment and stimulated protein kinase C enzymatic activity to a similar of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 K. R. Gustafson, J. H. Cardellina, II, J. B. McMahon, R. J. Gulakowski, M. ' To whom requests for reprints should be addressed. R. Boyd, J. Ishitoya, Z. Szallasi, N. E. Lewin, P. M. Blumberg, O. S. Weislow, 2The abbreviations used are: PKC, protein kinase C; PMA, phorbol 12- J. A. Beutler, G. M. Cragg, J. P. Bader, and P. A. Cox, Isolation and identification myristate 13-acetate; ODC, ornithine decarboxylase; PDBu, phorbol 12,13- of a new AlOS-antiviral agent from a Samoan medicinal plant, Homalanthus dibutyrate. acuminatus, submitted for publication. 5355

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1991 American Association for Cancer Research. PROSTRATIN extent as did PDBu.3 In C3H10T1/2 cells, it inhibited epider RESULTS mal growth factor binding and induced arachidonic acid release, Effects of Prostratin Pretreatment on PMA-induced Hyper typical phorbol ester responses, with 50% effective dose values of 2.2 x 10~7and 1.1 x 10~6 M, respectively (20-45-fold lower plasia. A dose of 16.2 nmol (10 Â¿ig)PMA induced strong hyperplasia on the back skin of CD-I mice at 48-72 h after potency than PDBu).3 In CD-I mice, a single prostratin treat treatment (Fig. 1.4). The hyperplasia could be inhibited dra ment induced edema and epidermal ornithine decarboxylase matically if the animals were pretreated with prostratin at an activity. The induction of ornithine decarboxylase showed a appropriate dose and an appropriate treatment schedule. The broader time course than with PMA, a maximal degree of inhibitory effect of 2.56 /Â¿mol(l mg) prostratin applied at 0 induction 25-30% that of PMA, and a maximal response at a and 48 h on the hyperplasia induced by 16.2 nmol PMA dose of 770 nmol (300 Â¿Â¿g)prostratin.3Of great interest, pros treatment 15 min after the second prostratin application is tratin did not induce hyperplasia in CD-I mice up to the highest illustrated in Fig. \B. dose examined, 2.56 /Â¿mol(l mg), with either a single treatment Within the severe limits imposed by the supply of prostratin, or with five treatments at 24- or 48-h intervals.3 In light of the the influence of the treatment conditions on the inhibition of correlation between chronic hyperplasia and tumor promoting the PMA-induced hyperplasia was examined. Using three ap activity (22), these results supported the earlier conclusion that plications of prostratin at 48-h intervals, followed by 16.2 nmol prostratin is not tumor promoting and suggested that further PMA at 48 h after the last prostratin application, 2.56 ^mol characterization of prostratin action might prove fruitful. prostratin were effective, but 256 nmol or lower doses failed to In the present report, we have examined the effect of single inhibit. Six applications of 2.56 /Â¿molprostratin at 48-h inter or multiple pretreatments of CD-I mouse skin with prostratin vals were likewise effective, whereas a single dose of 2.56 ÃŸmo\ on the subsequent response to PMA. The rationale was that, prostratin, followed by 16.2 nmol PMA at 15 min to 7 days since we did not obtain hyperplasia at prostratin doses that after the prostratin application, failed to inhibit hyperplasia. yielded acute responses, perhaps prostratin stimulated PKC Following two applications of prostratin separated by 48 h, pathways but also exerted a superimposed suppressive effect. hyperplasia was substantially inhibited for applications of 16.2 We have previously suggested an analogous explanation for the nmol PMA 15 min to 6 h after the last prostratin application, behavior of the bryostatins (6). We report here that prostratin was partially restored at 48 h, and had largely recovered by 96 pretreatment inhibits the response to PMA, with varying char h. When the interval between the two applications of 2.56 pmol acteristics for different responses. We conclude that prostratin, prostratin was varied, with challenge with 16.2 nmol PMA 15 although an in vitro activator of protein kinase C, represents a min after the last prostratin application, inhibition was most novel class of functional antagonists for protein kinase C me effective for intervals of 24 h to 8 days. diated responses. Because prostratin was more potent for inhibiting ODC induction than for blocking hyperplasia (see below), we also examined combinations of a lower dose of prostratin followed MATERIALS AND METHODS by a higher dose. Interestingly, although two applications of 256 nmol prostratin at an interval of 48 h failed to inhibit Female Charles River CD-I mice, 6-8 weeks of age, were obtained hyperplasia to 16.2 nmol PMA applied 15 min after the second from Charles River Laboratories, Wilmington, MA. The dorsal hair of application (Fig. 1C), the inhibition was complete for a first each mouse was shaved 2 days before starting the treatments, and only dose of 256 nmol prostratin, provided that the second dose was those mice showing no hair regrowth were used. All compounds were 2.56 Mmol (Fig. ID). dissolved in acetone and applied in a volume of 100 //I. Prostratin, PMA, and (â€”)-7-octylindolactam V were purchased from LC Services Since prostratin pretreatment blocked the hyperplasia ob (Woburn, MA). served in response to the usual dose of 16.2 nmol PMA we also examined its effect on a 10-fold higher dose of PMA (162 For analysis of ornithine decarboxylase activity, the epidermis of individual mice was separated from the dermis by brief heat treatment nmol). In control mice, 162 nmol PMA caused toxicity as (23). The epidermal preparations of two mice were pooled and then manifested by substantial necrosis in addition to hyperplasia. homogenized for 20 s at 0-4Â°Cin 50 mM sodium phosphate buffer, pH We found that 162 nmol PMA overcame the block of hyper 7.2, containing 0.1 HIMpyridoxal phosphate and 0.1 mM EDTA (23), plasia observed under the usual protocol of two pretreatments using a Polytron tissue homogenizer. The supernatant fraction obtained with 2.56 ÃÃmolprostratin; the necrosis, however, was largely after centrifugation at 30,000 x g for 2 x 20 min at O'C was used for prevented. We conclude that the inhibition of hyperplasia in determination of enzymatic activity, quantitated by the release of CO2 duced by PMA depended not only on the dose of prostratin but from [L-'4C]ornithine (Amersham, Arlington Heights, IL, and Dupont also on the dose of PMA evaluated. NEN, Boston, MA), as described by Lichti and Gottesman (24). In Effect of Prostratin Pretreatment on PMA-induced ODC Ac animals treated with solvent only, the CCh release was under 0.1 nmol tivity. The second response that we examined was induction of CO2/mg protein/h. ornithine decarboxylase. We had previously observed that a For measurement of edema, skins were removed after cervical dis single treatment with prostratin itself induced ODC, but the location, and 0.6-cm punches were cut out and quickly weighed. The skin punches were dried for 24 h at 60Â°Cand then reweighed. Data maximal induction was only 25-30% ofthat induced by 16.2 nmol PMA, and the peak of induction as a function of time were expressed as the ratio of the water content (wet - dry weight)/dry was broader.3 Maximal induction was observed at 770 nmol weight of each skin punch. (300 ng) prostratin3; the approximate 50% effective dose fell For examination of hyperplasia, dorsal skin was removed and fixed in 10% formalin in 0.1 M sodium phosphate buffer, pH 7.5. It was then between 70 and 770 nmol. We found that pretreatment with sectioned and stained with hematoxylin-eosin by American Histolabs, 256 nmol prostratin inhibited the induction of ODC by 16.2 Gaithersburg, MD. Under each set of conditions in each experiment, nmol PMA applied 48 h after the prostratin. Inhibition by a two animals were treated, two portions of the treated skin were excised single prostratin application was greater than 60%; that by two per animal, and three sections were prepared from each portion of the to six applications at 48-h intervals was 30-98% (Table 1). The skin. dose response curve for prostratin inhibition was determined 5356

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1991 American Association for Cancer Research. PROSTRATIN using three treatments at 48-h intervals. The 50% inhibitory Table 1 Effect of number of prostratin presentments on the ODC inducibility by PMA dose was 25.6 nmol (Fig. 2), markedly lower than the effective Four to six mice/group were treated with 256 nmol prostratin as many times dose for inhibition of hyperplasia and somewhat lower than as indicated at 48-h intervals. Forty-eight h after the last prelrcatment. 16.2 nmol that for the initial induction of ODC. Conversely, the time PMA were applied, and the ODC activity of preparations derived from two pooled skins was measured 6 h later. Each value represents the average Â±range or SEM. course for recovery from inhibition after three treatments with All compounds were applied in Kill i.l acetone. 256 nmol prostratin was slower than for inhibition of hyperpla activity induced sia (Fig. 3). Substantial inhibition remained at 4 days, and full protein)Experiment(nmol COj/h/mg No. of prostratin recovery was found at only 8 days. This rate of recovery is slow pretreatments0 11.76 24.59 compared to that of typical inhibitors. Â±0.13 Â±0.96 Since different agents can induce ODC with substantially 1 0.72 Â±0.15 0.56 Â±0.20 different time courses (25, 26), we checked whether there was 2 0.20 Â±0.02 0.50 + 0.16 3 0.25 Â±0.06 0.20 Â±0.09 any time shift caused by the prostratin pretreatment. Fig. 4 6ODC 0.21 Â±0.02Experiment0.18 Â±0.07 shows that the pretreated animals 3, 6, and 9 h after PMA treatment showed no significant elevation of ODC activity. To exclude the possibility that the inhibition by prostratin ^g) (-)-7-octylindolactam (4.9 Â±0.8 and 7.32 Â±3.89 nmol resulted from enhanced metabolism of PMA, we examined the CO2/h/mg protein in two independent experiments) was sev- effect on another potent PKC activator of a different structural class. (â€”)-7-Octylindolactam, a teleocidin analogue (27), lacks eralfold higher than that induced by 16.2 nmol PMA, but the ester residues of PMA which are cleaved during metabolic prostratin pretreatment again was inhibitory (1.11 Â±0.17 and breakdown (28). The ODC activity induced by 24.1 nmol (10 0.62 Â±0.04 nmol CO^/h/mg protein, respectively).

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Fig. 1. Effect of prostratin prclreatmcnt on the morphological changes of back skin induced by PMA. Mice were treated by the following protocols: (A) 100 nl acetone at 0 and 48 h and 16.2 nmol PMA 15 min later: (B) 2.56 nmol prostratin at 0 and 48 h and 16.2 nmol PMA 15 min later: (C) 256 nmol prostratin at 0 and 48 h and 16.2 nmol PMA 15 min later: (0) 256 nmol prostratin at 0 h. 2.56 Â¿imolprostratin at 48 h. and 16.2 nmol PMA 15 min later. Mice were sacrificed 72 h after the last treatment. All compounds were applied in 100 ÃŸ\acetone, x 400. Two more experiments yielded similar results. 5357

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1991 American Association for Cancer Research. PROSTRATIN over or other pathways producing elevated sn-1,2-diacylglycerol 4 -, levels (1,2). These signals play an important role in physiolog O ical regulation of almost all cells and are intimately involved in ^ O. the cancer process. Multiple oncogenes, including src, ras, and O) sis, elevate phosphatidylinositol turnover; transcription of cell 3 - E ular protooncogenes, including myc and fos, is mediated by IM O protein kinase C; protein kinase C regulates the activity of the Ãœ transcriptional activator protein c-jun and stimulates the mul- tidrug resistance system (29-32). Conversely, phorbol esters as _ Â£- O activators of protein kinase C might be potential differentiating E agents in cancer therapy (33). The conceptual obstacle to pro-

**o Â§Â°o o W 2.56 7.68 25.6 76.8 256 768 2560 o. O) dose of prostratiti pretreatment {nmol) E ^ Fig. 2. Dose dependent inhibition of PMA induced ODC activity by multiple

140 -i u Â« Ãœ O 3 6 O time after PMA treatment (hrs) Fig. 4. Time dependence of the induction of ODC activity by PMA after prostratin or solvent-only pretreatment. Six mice/group were treated with 256 nmol prostratin (â€¢)orsolvent only (D) three times at 48-h intervals. Forty-eight h after the last pretreatment. 16.2 nmol PMA were applied to the back skin, and the ODC activity of preparations derived from pooled skins was measured 3. 6, and 9 h later. Each value represents the average Â±SEM. All compounds were applied in 100 <

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ra time after the last prostratin treatment (days) E 5 - Fig. ÃŒ.Recoveryof ODC inducibility after prostratin pretreatment. Six mice/ group were treated with 256 nmol prostratin (â€¢)three times at 48-h intervals. 4 Fifteen min, 6, 12. and 24 h. and 2, 4. and 8 days after the last pretreatment. 16.2 nmol PMA were applied to the back skin, and the ODC activity of prepa rations derived from two pooled skins was measured 6 h later. The results were 3 - expressed as percentages of the control values derived from mice pretreated with O soivent only. Data representing the same time points in three independent O experiments were pooled. Each value represents the average Â±SEM. All com 2 - pounds were applied in 100 ii\ acetone. m i 1 - Effect of Prostratin Pretreatment on PMA-induced Back Skin Edema. Prostratin pretreatment reduced the level of edema induced by 16.2 nmol PMA in a dose dependent manner (Fig. 76.8 256 768 2560 5). Because of limitations on the amount of prostratin, we were not able to determine whether we could achieve complete inhi dose of prostratin pretreatment (nmol) bition if the dose were further increased beyond 2.56 //mol. Fig. 5. Dose dependent reduction of PMA induced edema by multiple prostra tin pretreatments. Six mice/group were treated with the indicated doses of prostratin three times at 48-h intervals. Forty-eight h after the last pretreatment, 16.2 nmol PMA (â€¢)or solvent (D) was applied to the back skin, and 6 h later, DISCUSSION skin punches of 0.6-cm diameter were removed. Edema is expressed as the ratio of the water content/dry mass, where the water content is the difference between Protein kinase C occupies a central role in transduction of the wet and dry weight. Each point represents the average of six animals Â±SEM. signals generated through enhanced phosphatidylinositol turn A second experiment yielded similar results. 5358

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1991 American Association for Cancer Research. PROSTRATI N tein kinase C as a therapeutic target has been the diversity of the ability of a low dose of prostratin to potentiate inhibition its biological roles and the need for greater selectivity. of hyperplasia by a single high dose of prostratin. The concept Most of the current inhibitors of protein kinase C, such as is further supported by the biphasic effect of prostratin on p24 H7 (34), sphingosine (35), and staurosporine (36-39), are trou viral antigen production and reverse transcriptase activity in bled by their low selectivity. The most noteworthy exceptions CEM-SS cells and on the syncytium formation in both CEM- are the staurosporine analogues (40) and the partial functional SS and C-8166 cells which we observed earlier.-' Mechanistic antagonist bryostatin (6). The former are candidates for inter understanding may further the rationale design of therapeutic acting at the ATP site in the catalytic domain of the enzyme. modulators of protein kinase C. The latter binds specifically to the regulatory domain, activates the enzyme, but anomalously blocks some biological responses. ACKNOWLEDGMENTS Our results presented here demonstrate that prostratin resem bles bryostatin in blocking select protein kinase C mediated We thank Dr. Stuart H. Yuspa and Dr. Henry Hennings for their responses, although, like bryostatin, it activates protein kinase critical reading of the manuscript. C in vitro. Prostratin differs from other classes of functional inhibitors of protein kinase C in the need for chronic pretreat REFERENCES ment. Since phorbol ester pretreatment can lead to protein kinase C down-regulation and loss of responsiveness in biolog 1. Nishizuka. Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1991 American Association for Cancer Research. Prostratin, a Nonpromoting Phorbol Ester, Inhibits Induction by Phorbol 12-Myristate 13-Acetate of Ornithine Decarboxylase, Edema, and Hyperplasia in CD-1 Mouse Skin

Zoltan Szallasi and Peter M. Blumberg

Cancer Res 1991;51:5355-5360.

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