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).

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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 purified by lysmne-Sephamose Schwarz/Mann, Orangebung, N. Y. Dexamethasone, con affinity chromatography (7). Aliquots were taken after 4 to 8 texolone, cortisol, testosterone, estnadiol, and 7,12-dimeth hr, and the solubilized fibnin was determined by scintillation ylbenz(a)anthmacene were purchased from Sigma Chemical counting in Aquasol using the 3H channel. The percentage Co., St. Louis, Mo. TPA was prepared as previously de of fibnin digested was determined from the ratio of the scnibed (1) and purified by preparative thin-layer chroma counts released in the presence of sample (adjusted for togmaphy. The antiinflammatony steroids fluocinonide fibnin released in the presence of plasminogen but in the (6a,9a-difluoro-1i f3,16a,1 7,21 -tetrahydroxypregnan-i ,4- absence of sample) to the total counts released by trypsmn. diene-3,20-dione, cyclic 16,17-acetal, 21-acetate), FA, and Fibninolysis was negligible in the absence of plasminogen FCA were a gift from the Syntex Corp. , Palo Alto, Calif. The (39). nonsteroidal antiinflammatomy agents were generously sup plied as follows: oxyphenbutazone [Tandeanil; 1-phenyl-2- (p-hydroxyphenyl)-3,5-dioxo-4-n-butylpynazolidine mono RESULTS hydrate] from Geigy Pharmaceuticals, Ardsley, N. Y.; Se clazone [W-2354; 7-chloro-3,3a-dihydmo-2H,9H-isoxa Effects on Tumor Promotion. In view of the previously zolo(d,2-b)-(1 ,3)-benzoxazin-9-one] from Wallace Labonato reported inhibitory effects of dexamethasone (23), we as nies, Cranbury, N. J.; and indomethacin [i-(p-chloroben sayed several antiinflammatony agents for their ability to zoyl)-5-methoxy-2-methylmndole-3-acetic acid] Sharp and inhibit the promotion phase of 2-stage skin tumonigenesis in Dohme, Rahway, N. J. mice given a single initiating dose of 7,12-dimethyl (3HjdThd Incorporation into DNA. Mice were killed 30 mm benz(a)antracene (51 @g)and then twice weekly topical after i.p. injection of 30 @Ciof [3H]dThd. All experiments application of the promoting agent TPA. Two types of an were performed between 8 a.m. and 1 p.m. to minimize the tiinflammatony agents were used in this study in order better effects of diurnal variation. The epidermis was isolated as to understand the role that inflammation plays in tumor

MAY 1977 1S31

promotion and chemical carcinogenesis in general. First, thesis was followed by an actual stimulation at 10 days after other antiinflammatory steroids known to have antiinflam theinitial FA treatment. Histologically, FA did not cause any matory activity when given either topically or systemically observable ulceration or loss of epidermal cell layers, but it (18)@were used. Second, nonstenoidal antiinflammatory did cause some thinning of the dermis and some subtle agents that are active in suppressing development of adju effects on the epidermal cells. Table 2 shows the effects of vant-induced polyarthnitis in rats when these agents were several add itional anti inflammatory agents on epidermal given p.o. or systemically and that also counteract hind paw DNA synthesis. It is apparent that fluocinonide and FCA phlogistic response induced in rats by irritating agents (18) were also potent inhibitors of epidenmal DNA synthesis. A were used . The above response is the classical method for high dose (1 mg) of oxyphenbutazone had a slight inhibitory determining antiinflammatony ability of compounds (37, 40). effect on DNA synthesis at 12 and 18 hr after topical treat Also, the effects of the nonsteroidal agents used do not ment, whereas the same dose of Seclazone or indometha appear to be mediated through the adrenal-pituitary axis. cm had no appreciable effect. The antiinflammatory steroids FA, fluocinonide, and FCA The results of topical applications of various antiinflam were found to be potent inhibitors of tumor promotion in matory agents on TPA-induced inflammation and epidermal mouse skin when administered topically at 10 j.@g/0.2ml cell proliferation are shown in Table 3. When FA, FCA, or simultaneous with each exposure to TPA (Chart 1). Similar fluocinonide were applied simultaneously with TPA in 1O-@g degrees of inhibition were noted; however, the data suggest doses, they completely counteracted the TPA-induced in that tumor latency was increased most with FA. In previous crease in nucleated interfollicular epidenmal cell layers, in experiments (23), the doses of dexamethasone required to flammation, and edema. The same dose of dexamethasone cause marked suppression of TPA promotion were 50 j.@g/ was slightly less effective, whereas the nonsteroidal antiin application and the ratio of dexamethasone to TPA concen flammatory agents had either no effect or only a slight effect tration was 50. In Chart 1 only [email protected] of the new on TPA-induced inflammation and cellular proliferation. synthetic antiinflammatory steroids (a ratio of steroid to TPA Effects on Plasminogen Activator Production in Cell Cul concentration of 5) were sufficient to produce a comparable ture. Table 4 shows the inhibitory effect of various antiin suppression. Thus, in this respect, these compounds are 10 flammatory steroids on the production of plasminogen acti times more potent than dexamethasone. vator by cell cultures of the HTC cell line. FA and FCA, in Table 1 compares the effects of the nonsteroidal antiin concentrations as low as 0.5 ng/mI (i0@ M), caused greater flammatory agents oxyphenbutazone, indomethacin, and than 95% inhibition of plasminogen activator production. Seclazone to that of FA in terms of inhibition of TPA-related Additional studies indicated that even 10â€• M FCA or FA tumor promotion in mouse skin. One-mg doses of oxyphen produced appreciable inhibition. A dexamethasone con butazone or Seclazone, given topically together with TPA, centration of about 0.045 j.@g/ml(10@M) and a cortexolone caused a slight inhibition of tumor promotion. Idomethacin concentration of about 3.5 @g/ml(10@M) were required to was toxic at repeated topical doses greater than 100 j.@g;at produce a greater than 95% inhibition. Thus, the order of 50 pg/dose it caused only a slight inhibition of tumor pro potency of these glucocorticoids with respect to this effect motion (Table 1). For avoidance of the local skin toxicity, is FA = FCA > dexamethasone > cortexolone. This order additional studies were done in which the test compound correlates with the relative potency of these compounds as was given i.p. ratherthan topically. High (1-mg) i.p. doses of antiinflammatony agents.4 Separate studies have shown that oxyphenbutazone resulted in a 58% inhibition, and i-mg testosterone on estradiol at i0@ M did not inhibit plasmino i.p. doses of indomethacin resulted in a 50% inhibition of gen activator production (38). the average number of papillomas resulting from TPA-ne Iated promotion. In separate studies not shown here, we 100 have also found that topical application of oxyphenbuta @;80- @ zone inhibited the initiating ability of MC. Table 1 again 60- L&JO demonstrates the marked potency of FA as an inhibitor of @:J40 @ 20@ tumor promotion. The comparison of nonstenoidal to steroi 0.0 dal antiinflammatory agents could be misleading because B of the possible lack of percutaneous absorption in vivo of Lii 6 p0 the nonsteroidal agents. U) Op Effects on [3H]dThd Incorporation, Inflammation, and 4. pd Epidermal Cell Proliferation. The effect of a [email protected] U) 2 FA on the incorporation of [3H]dThd into DNA of normal 9 mouse epidermis is depicted in Chart 2. Within 1 hr after topical application of FA to the skin of mice, epidermal DNA synthesis was drastically inhibited and the inhibition lasted WEEKS OF PROMOTION for approximately 6 days. A greater than 80% inhibition of Chart 1. Inhibition of tumor promotion by simultaneous treatment with DNA synthesis was observed from Days 1 to 5 following FA TPA plus either FA, FCA, or fluocinonide. Each group consisted of 30 mice, and all mice were initiated with 200 nmoles (51.2 @g)of 7,12-dimethyl treatment. This prolonged inhibition of epidermal DNA syn benz(a)anthracene and promoted with 2 @gof TPA given twice weekly starting 1 week after initiation: A, percentage of mice with papillomas as a function of weeks of promotion; B, average number of papillomas per mouse as a function of weeks of promotion. 0, control (TPA alone); â€¢,TPA 4 Syntex Corp. , personal communication on the relative antiinflammatory plus 10 lLg of fluocinonide; & TPA plus 10 @gofFCA; A, TPA plus 10 tg of potencies of FA, fluocinonide, and FCA. FA.

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Table 1 Inhibition of TPA tumor promotionoxyphenbutazone,indomethacin, in mouse skin by topical treatment with FAAll Seclazone, and bytwice-weeklymice were treated once with 200 nmoles of 7,12-dimethylbenz(a)anthracene followed treatment with 2 @gofTPA. Each group consistedmice.% of 30 papillo- Time to Av. papillo mas/mouseGroup mas at Wk 1st tumor 241 ModifierÂ° Dose(pg) 24 (wk) at Wk 8.52 Acetone 82 6 5.03 Oxyphenbutazone 1000 65 7 6.84 Indomethacin 50 73 6 6.05 Seclazone 1000 60 6 FA 10 7 >40 0.1

(I The modifiers were applied simultaneously with TPA at the dose indicated. Acetone is the solvent control.

@ a, owed the order fluocinonide FA > FCA > dexametha @ 2OO@ sone > cortisol. It is remarkable that the compounds fluoci C 0 â€˜a nonide, FA, and FCA were active on mouse skin at dosages as low as a few j.@gand,in cell culture, at concentrations as >- @ I- low as M. In this study, we also examined the nonster > oidal antimnflammatory agents oxyphenbutazone, indo methacin, and Seclazone, since these agents (when given U Li systemically) inhibit experimentally induced inflammation U Lii 0. in animal systems (37, 40) and inflammatory diseases in U) man (18). We found, however, that in general the effects of 0 2 4 6 8 10 these agents on TPA-induced inflammation, tumor promo DAYSAFTERFA TREATMENT tion, and DNA synthesis in mouse skin (when given topi Chart 2. The effect of a single topical application of 10 @tgofFA on the cally), and on plasminogen activator production in cell cul incorporation of [3H]dThd into mouse epidermal DNA. Incorporation time for [3H]dThd was 30 mm. The specific activity results for each time point are ture, were not very impressive when compared to the pow expressed as percentage of average specific activity of the acetone (control) erful inhibitory effects of the steroidal agents. mice. Each time point represents an average of 2 to 3 groups containing 3 mice each. - - - -, standard deviation for the mice in the acetone (control) Since the precise mechanism of action of TPA and related group. The average specific activity for the acetone controls was 25 dpm/j.@g promoting agents is not known, any discussion of the of DNA. mechanism of inhibition of tumor promotion by steroidal agents must at the present time be largely speculative. It has recently been emphasized that phorbol ester promotors Table S indicates the effects of the nonstenoidal antiin induce several biological effects, in mouse skin or in cell flammatory agents on plasminogen activator production. cultures, that mimic the phenotypes of cells transformed by Oxyphenbutazone at 4 @.tg/mlproduced about a 50% inhibi viral on chemical carcinogens (39). These include: (a) epi tion, and at 40 @g/mlit produced about a 92% inhibition. dermal hyperplasia in mouse skin and increased saturation Indomethacin was less active, since 4 @g/mlproduced density of certain cell cultures (28, 39); (b) altered cell about a 22% inhibition and 40 j.tg/ml gave approximately a morphology (20); (C) increased phospholipid synthesis (22, 42% inhibition. Similar concentrations of Seclazone pro 30, 31); (d) induction of onnithine decarboxylase (16, 41); duced at most a slight inhibition. Thus, indomethacin and (e) induction of DNA synthesis (41); (f) decreased cellular oxyphenbutazone can inhibit plasminogen activator pro cyclic adenosine 3':S'-monophosphate levels (4); (g) induc duction, but they are much les potent than were dexameth tion of plasminogen activator production (39); and (h) cell asone, FCA, or FA. transformation by chemical carcinogens in cell culture (13). The marked induction of plasminogen activator produc DISCUSSION tion is of particular interest since a variety of studies on protease production associated with cell proliferation (5, This report, as well as previous studies (3, 15, 23), mdi ii), cell transformation (6, 10, 17, 36), and tumonigenesis cates that the antimnflammatony steroids are extremely ac (6, 19, 35) suggest that plasminogen activator production tive inhibitors of the tumor-promoting phonbol esters. We may be an important aspect of the process of cancinogene have also demonstrated that the relative antimnflammatory sis. A relationship has also been demonstrated between potencies of a series of synthetic fluorinated glucocorti production of plasminogen activator and various features of coids correlate with their ability (a) to inhibit mouse skin the transformed phenotype, such as cell locomotion, mom tumor promotion by TPA, (b) to counteract TPA-induced phology, and loss of anchorage-dependent growth (17) in skin hyperplasia, (c) to inhibit epidermal DNA synthesis, cell culture. On the other hand, several exceptions to this and (d) to inhibit plasminogen activator production in cell association have been reported (12, 14). It is not clean, culture. The relative potencies of the steroids tested fol therefore, whether the production of plasminogen activator

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1977 American Association for Cancer Research. A. Viaje et at. Table 2 The effects of antiinflammatory agents on the incorporation of (3HjdThd into epiderma! DNA Eachvalue[3H]dThd, 30 @Ci(6Ci/mmole), was injected 30 mm before animals were killed. was obtained from pooled skins of 3 or 4mice.Specific atfollowingactivityaof DNA(%of controls) treatmentAntiinflammatory times after

hrFluocinonide agent Dose (pg) 12 hr 18 hr 24 hr 48 7FCA 10 18 6 4 12Oxyphenbutazone 10 29 20 15 104Seclazone 1000 70 81 108 112Indomethacin 1000 94 108 97 1000 83 86 92 80

(1 Specific activity was calculated as dpm/@.@g DNA and expressed as percentage of the valueobtainedwith control mice.Theaveragespecificactivity for the acetonecontrol was 22.2 dpm/@gDNA.

3Effects Table of various topicallyinflammationand applied antiinflammatory agents on TPA-induced proliferationThe epidermal cellular andedematousnumber of nucleatedinterfollicular epidermalcell layersand the inflammatory themean indices were determinedin 5-pm sections of skin. Eachvalue represents detailsseeof 7 observations.The S.D. was lessthan 20%of eachvalue. For additional Methods.â€•AMaterials and epidermalcellularprimary treatment of 10 @gofTPAwas given to induce inflammation and insteadof proliferation in all cases except for the 1 (control) entry for which acetone, TPA, was used.Nucleatedintertollicularepidermal

Inflammatory in dexbSecondary-treatmenr cell layers Edemaantiinflammatory indexdNone agent Dose (pg) 3c 4C le 2C 0None(control) 1.5 1.3 0 0 4FA 4.6 5.5 4 4 0FCA 10 1.4 1.2 0 0 0Fluocinonide 10 1.7 1.5 1 0 0Dexamethasone 10 1.6 1.3 0 0 1Oxyphenbutazone 10 2.2 2.4 1 1 3Indomethacin 1000 4.0 4.2 3 3 3Seclazone 1000 4.2 4.8 3 4 1000 4.7 5.4 4 4 4 a Application of the antiintlammatory agent simultaneous with TPA. b The degree of inflammation was classified from 1 to 4, with Grade 4 indicating maximal responseand zero indicating no effect.

C Days posttreatment. (I The degree of edema was classified (1 day after treatment) from 1 to 4, with Grade 4 representing maximal responseand zero representingno effect.

is causally related to the other aspects of the tumor cell activator in mouse epidermal cells in culture and in epider phenotype on is merely 1 of a number of pleiotnopic mani mis in ViVO.We are also determining whether glucocorti festations of a more fundamental derangement in growth coids can counteract plasminogen activation in the above control. Further studies are required, therefore, to establish more relevant system. whether or not the ability of certin steroids to inhibit the Regardless of the precise underlying mechanism, the me process of tumor promotion relates to their ability to inhibit suits obtained with highly potent synthetic steroids lend plasminogen activator production (38) or whether inhibition encouragement to further explorations of the important of plasminogen activator production is simply a convenient area of inhibition of specific stages of the process of carci marker of a more basic mechanism by which glucocorti nogenesis. This is a somewhat neglected area of research coids inhibit tumor promotion. Studies are under way to that has obvious relevance to the problem of human cancer determine whether phorbol esters will induce plasminogen prevention.

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Table 4 Skin Tumorigenesisby Steroid Hormones.Cancer Res.,32: 450-454, SteroidcellculturesCells inhibition of plasminogenactivator production in HTC 1972. 4. Belman, S., and Troll, W. Phorbol-12-Myristate-13-Acetate Effect on Cyclic Adenosine 3',5'-Monophosphate Levels in Mouse Skin and Inhibi testagentwere pretreated with the indicated concentration of the tion of Phorbel-Myristate-Acetate-promoted Tumongenesis. Cancer releaseoffor 24hr. Lysateswerethen preparedand assayedfor Res., 34: 3446-3455, 1974. ofplasminogenradioactivity from â€˜2l-labeledfibrinplates in the presence 5. Burger, M. M. Proteolytic Enzymes Initiating Cell Division and Escape assayswere as described In â€œMaterialsandMethods.â€•All from Contact Inhibition of Growth. Nature, 227: 170-171, 1970. 20%.Releaseperformed in triplicate, and replicates agreed within 6. Christman, J. K., Acs, G., Silagi, S., and Silverstein, S. C. Plasminogen Activator: Biochemical Characterization and Correlation with Tumori orplasminogenof â€˜2l-labeled fibnin in the absence of either lysate genicity. In: E. Reich, D. B. Rifkin, and E. Shaw (eds.), Proteases and was negligible.â€˜25l-labeled Biological Control, pp. 827-839. Cold Spring Harbor, N. Y.: Cold Spring digestedPretreatment fibnin Harbor Laboratory, 1975. 7. Deutsch, D. G., and Mertz, E. T. Plasminogen: Purification from Human Plasma by Affinity Chromatography. Science, 170: 1095-1096, 1970. of Concentra- % of con 8. Frei, J. V., and Stephens, P. The Correlation of Promotion of Tumor trolNoneHTCcells tion (M) % digested Growth and Induction of Hyperplasia in Epidermal Two-State Carcino 100FCA 21.2 genesis. Brit. J. Cancer, 22: 83-92. 1968. 9. Ghadially, R. N., and Green, H. N. The Effect of Cortisone on Chemical Carcinogenesis in the Mouse Skin. Brit. J. Cancer, 8: 291-295, 1954. 10@410-i 0.9 10. Goldberg, A. R., wolf, B. A., and Lefebure, P. A. Plasminogen Activators 510-i 1.0 ofTransformed and Normal Cells. In: E. Reich, D. B. Rifkin, and E. Shaw 5FA 1.0 (eds.), Proteases and Biological Control, pp. 857-868. Cold Spring Har bor, N. V.: Cold Spring Harbor Laboratory, 1975. 10@310-s 0.7 11. Hennings, H., and Boutwell, R. K. Studies on the Mechanism of Skin 410-p 0.9 Tumor Promotion. Cancer Res., 30: 312-320, 1970. 4Dexamethasone 0.9 12. Laug, W. E., Jones, P. A., and Benedict, W. F. Studies on the Relation ship between Fibrinolysis of Cultured Cells and Malignancy. J. NatI. Cancer Inst., 54: 173-179, 1975. 10'7710â€”i 16.3 13. Mondal, S., Peterson, A. R., and Brankow, D. W. Initiation and Promo 1110-' 2.3 tion in Oncogenesis in Cell Cultures. Proc. Am. Assoc. Cancer Res., 16: 410-i 0.9 74.1975. 3Cortexolone 0.6 14. Mott, D. M., Fabisch, P. H., Sani, B. P., and @orof,S.Lack of Correlation between Fibrinolysis and the Transformed State of Cultured Mammalian 10@7610-i 16.2 Cells. Biochem. Biophys. Res. Commun., 61: 621-627, 1974. 6810-s 14.4 15. Nakai, T. Influences of Small Doses of Various Corticosteroids on the Incidence of Chemically Induced Subcutaneous Sarcomas in Mice. Can 5110@ 10.8 cer Res., 21: 221-227, 1961. 4.9 23 16. O'Brien, T. G., Simsiman, R. C., and Boutwell, R. K. Induction of the Polyamine-biosynthetic Enzymes in Mouse Epidermis by Tumor-promot ing Agents. Cancer Res., 35: 1662-1670, 1975. 17. Ossowski, L., Quigley, J. P., Kellerman, G. M., and Reich, E. Fibrinolysis Table 5 Associated with Oncogenic Transformation. J. Exptl. Med., 138: 1056- 1064,1973. Inhibition ofplasminogen activatorproduction in HTCcell cultures 18. Physicians' Desk Reference, Ed. 30. Oradell, N.J.: Medical Economics by various nonsteroidal antiinflammatory agents Corp., 1976. Cells were pretrea with the testcompound and lysates as 19. Pollack, R., Risser, R., Colon, S., and Rifkin, D. Plasminogen Activator sayed as in Table 4.ted Production Accompanies Loss of Anchorage Regulation in Transforma tion of Primary Rat Embryo Cells by Simian Virus 40. Proc. NatI. Acad. fibrin di Sd. U. 5.,71: 4792-4796, 1974. gested% 20. Raick, A. N. Ultrastructural, Histological, and Biochemical Alterations Produced by 12-O-Tetradecanoylphorbol-13-acetate on Mouse Epider Pretreatment of con mis and Their Relevance to Skin Tumor Promotion. Cancer Res., 33: trolControl18.8of HTCcellsConcentration (@tg/ml)125l@Iabeled% digested 269-286, 1973. 21. Raick, A. N., and Burdzy, K. Ultrastructural and Biochemical Changes 100Dexamethasone Induced in Mouse Epidermis by a Hyperplasia Agent, Ethylphenylpropi olate. Cancer Res., 34: 920-926, 1974. 5.32Oxyphenbutazone4 .0 22. Rohrschneider, L. R., O'Brien, D. H., and Boutwell, R. K. The Stimula (10' M)0.451 tion of Phospholipid Metabolism in Mouse Skin following Phorbol Ester Treatment. Biochim. Biophys. Acta, 280: 57-70, 1972. 23. Scribner, J. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1977 American Association for Cancer Research. Effects of Antiinflammatory Agents on Mouse Skin Tumor Promotion, Epidermal DNA Synthesis, Phorbol Esterinduced Cellular Proliferation, and Production of Plasminogen Activator

Aurora Viaje, Thomas J. Slaga, Michael Wigler, et al.

Cancer Res 1977;37:1530-1536.

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