Effects of Antiinflammatory Agents on Mouse Skin Tumor Induced Cellular
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[CANCER RESEARCH 37,1530-1536,May1977] Effects of Antiinflammatory Agents on Mouse Skin Tumor Promotion, Epidermal DNA Synthesis, Phorbol Ester induced Cellular Proliferation, and Production of Plasminogen Activator1 Aurora Viaje, Thomas J. Slaga,2 Michael Wigler, and I. Bernard Weinstein Cancerand ToxicologyProgram,Biology Division, OakRidgeNationalLaboratory,OakRidge, Tennessee37830(A. V., T. J. S.J,and Institute of Cancer ResearchandDepartmentsof Microbiologyand Medicine,ColumbiaUniversity,Collegeof PhysiciansandSurgeons,NewYork,New York 10032(M. W.,I. B. W.) SUMMARY Schening No. 11572 > prednisolone > hydmocortisone > cortisone) correlated with their antiinflammatomy activities The antiinflammatory steroids fluocinolone acetonide, in mouse skin. These investigators also showed that dexa fluocinonide, and fluclonolone acetonide were found to be methasone inhibited croton oil-induced epidenmal hyper very effective inhibitory agents of mouse skin tumor promo plasia. Cortisone has also been shown to inhibit complete tion. These steroids also drastically inhibited epidemmal carcinogenesis in mouse skin (9). In separate studies, Nakai DNA synthesis and epidermal cellular proliferation induced (15) demonstrated that the Induction of s.c. sarcomas in by a phonbal ester tumor promoter. In addition, these com mice by MG3was also inhibited by steroids in the following pounds were potent inhibitors of plasminogen activator order: dexamethasone > triamcInolone > methylpredmniso production in tumor cell cultures. The clinically used non lone > hydrocortisone > cortisone. This effect also come steroidal antiinflammatory agents oxyphenbutazone, indo lated with the antimnflammatory potencies of the steroids. methacin, and Seclazone also inhibited tumor promotion Subsequent studies by Scnibner and Slaga (23) showed that but were much less effective. Although these agents are appropriate does of dexamethasone completely suppressed useful against inflammatory disorders in general when tumor promotion in mouse skin by TPA for at least 6 given p.o., in our studies they had little effect on inflamma months. Dexamethasone was further found to reduce tumor tion and epidermal cellular proliferation induced by a phor initiation in mouse skin and carcinogenesis induced by MC bol ester tumor promoter when given topically. The afore alone (26, 33). In addition it inhibited mouse epidermal DNA mentioned nonsteroidal antiinflammatory agents also had synthesis (29) and reduced several protein fractions (on little effect on epidermal DNA synthesis. Oxyphenbutazone polyacrylamide gels) that were greatly enhanced after TPA and indomethacin were less potent inhibitors of plasmino treatment (23, 27). gen activator production in tumor cells than were the antiin Whereas most skin tumor promoters (phombol esters) ap flammatory steroids, and Seclazone produced a negligible pear to be irritants and induce epidermal hypemplasia (1, 8, inhibition. There is, therefore, a general correlation in the 20), not all irritants, inflammatory agents, on hyperplastic potencies of a series of steroidal antimnflammatory agents agents are promoters (11, 21, 24). However, the latter state for inhibition of tumor promotion and their ability to inhibit ment should be qualified, since most non-phorbol ester plasmmnogen activator production by tumor cell cultures hyperplastic agents do possess very weak promoting activi and epidenmal DNA synthesis. ties (21, 24). A correlation was observed between the skin tumor-promoting abilities of a series of phorbol esters and their ability to induce a sustained stimulation of RNA, pro INTRODUCTION tein, and DNA synthesis in mouse epidermis (2). A comrela tion was also found between the amount of phonbol-12,i3- Belman and Troll (3) showed that a series of steroidal dioctanoate that produced the maximum promoting effect antiinflammatory agents inhibited tumor promotion by cr0- and that which caused maximum induction of epidermal ton oil in mouse skin in a dose-dependent manner. The hyperplasia (28). Slaga et at. (28) also found a good comrela relative potency of these compounds (dexamethasone > tion between the promoting ability of various phonbol esters and their ability to induce epidermal hyperplasia. 1 This research was supported in part by NIH Grants CA-02332 and CA Wiglen et at. (38) have recently found that extremely low 17605, NCI Contract 72-3234, and the Energy Research and Development Administration under contract with Union Carbide Corporation. By accept ance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any 3 The abbreviations used are: MC, 3-methylcholanthrene; TPA, 12-0-tetra copyright covering the article. decanoyl-phorbol-13-acetate; [3H]dThd, tritiated thymidine; FA, fluocinolone 2 To whom requests for reprints should be addressed, at Biology Divi acetonide (6a, 9a-difluoro-1 1f3,16a,17,21-tetrahydroxypregnan-1 ,4-diene sion, Oak Ridge National Laboratory, Post Office Box V Oak Ridge, Tenn. 3,20-dione, cyclic 16,17-acetal); FCA, fluclorolone acetonide (6a-fluoro 37830. 9a,11fl-dichloro-16a,17a,21-triolpregnan-1,4-diene-3,20-dione,cyclic16,17- Received November 30, 1976; accepted February 15, 1977. acetal). 1@30 CANCER RESEARCH VOL. 37 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1977 American Association for Cancer Research. Antiinftammatory Agents and Tumorigenesis concentrations of dexamethasone and related glucocorti previously described (25). DNA hydrolysates were prepared coids produce a marked inhibition of plasminogen activator from the epidermis after the material was pooled from production in certain tumor cell cultures. On'the other groups of 4 mice by a modified Schmidt-Thannhauser pro hand, TPA proved to be a potent inducer of plasminogen cedune (25). The specific activity of the DNA was expressed activator in other cell cultures (39). These results, as well as as dpm/@tg of DNA and as a percentage of the values ob a variety of studies on protease production associated with tamed with the control group. cell proliferation, tumonigenesis, and cell transformation, Inflammation and Epidermal Cellular Proliferation. The suggest that an important aspect of skin tumor promotion number of nucleated interflollicular epidermal cell layers may be the production of specific proteases (5, 10, 12, 17, and the number of inflammatory cells in the dermis were 18, 34-36, 39). The ability of certain glococorticoids to measured in 5-gm sections of skin stained with hematoxylmn inhibit both skin tumor promotion and plasminogen activa and eosin by a modification (28) of the procedure described ton production provides indirect support for this hypothesis. by Raick (20). Ten-pg doses of TPA were used to induce This report describes studies on the effects of both steroi inflammation and hyperplasia. The effects of the antiinf lam dal and nonsteroidal antiinflammatory agents on mouse matory agents on TPA-induced cellular proliferation were skin tumor promotion, epidemmal DNA synthesis, and TPA determined by applying the antiinflammatomy agent simulta induced epidermal hyperplasia and inflammation. These neously with the TPA. same agents have been studied for their ability to inhibit Cell Cultures. The HTC cell line, originally established plasminogen activator production in tumor cell cultures. from a rat hapatoma, was obtained from Brad Thompson (32). Cells were grown as monolayer cultures in plastic Petmi dishes and were fed 2 on 3 times weekly with Ham's F-i2 MATERIALS AND METHODS medium + 10% fetal calf serum. Cells were passaged upon reaching confluence. Animals. Female Charles River CD-i mice were pun Plasminogen Activator Assays. Assaysfor plasminogen chased from Charles River Mouse Farms, North Wilming activator were performed by a slight modification of a previ ton, Mass. Seven- to 9-week-old mice were carefully shaved ously published procedure (36). The assay was based on with surgical clippers 2 days before treatment, and only quantitation of the amount of 125l-labeled fibnin solubilized those mice in the resting phase of the hair cycle were used by proteolytic digestion in the presence of plasminogen and in the biochemical and tumor experiments; in the tumor sample. Confluent plates of HTC cells (approximately 5 x experiments, groups of 30 mice were used. The incidence 106 cells per 10-cm dish) were mefedwith growth medium of both papillomas and carcinomas was recorded weekly, containing test agents at various concentrations. After 24 and a random sample of these was taken for histological hn, cells were washed twice with phosphate-buffered saline verification. The antiinflammatomy agents were given to and collected by scraping into 2 ml of swelling buffer (10 gether with each topical application of TPA. We found simi mM Tnis:10 mM NaCI, pH 8.0) without detergents. The sus Ian results when the antiinflammatory agents were given 30 pension was frozen and thawed 3 times and mixed vigor mm before each TPA application. In some cases, the antiin ously. This crude lysate was then diluted 1:10 into assay flammatony agents were administered i.p. 5 mm prior to buffer (100 mM Tnis:10 mri NaCI, pH 8.0) and assayed in topical application of TPA. Additional details of the animal triplicate as described by Unkeless et at. (36) on fibnin procedures were as previously described (24). coated plates in the presence or absence of human plas Chemicals. [3H]dThd (6.0 Ci/nmole) was obtained from mmnogen, 4 @g/ml,previously