Structure-Activity Relationships Among Various Retinoids and Their
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[CANCER RESEARCH 40. 3141-3146, September1980] 0008-54 72/ 80/ 0040-0000$02.00 Structure-Activity Relationships among Various Retinoids and Their Ability to Inhibit Neoplastic Transformation and to Increase Cell Adhesion in the C3H/10TV2 CL8 Cell Line1 John S. Bertram Department of Experimental Therapeutics, Grace Cancer Drug Center. Roswell Park Memorial Institute, Buffalo, New York 14263 ABSTRACT these observations, we have examined the effects of retinoic acid itself and a series of synthetic analogs (21, 3) for their Various natural and synthetic retinoids have been studied for ability to inhibit MCA-induced transformation and to cause their activity in two biological systems: (a) their activity as increased adhesion of fibroblasts to a plastic substrate. inhibitors of methylcholanthrene-induced neoplastic transfor This latter parameter was studied because of the recent mation in the C3H/1 OT'/2clone 8 mouse fibroblast line (System report of a correlation between the activity of retinoic acid and 1); and (to) their ability to increase the degree of adhesion of certain analogs in causing increased adhesion of BALB/3T12 C3H/10T'/2 clone 8 cells to a plastic substrate (System 2). cells and their activity in maintaining epithelial differentiation These activities were then compared with their known activity (1, 10). The results presented here suggest that no general in maintaining epithelial differentiation (System 3). With the notable exception of retinoic acid and 13-c/'s-retinoic acid, conclusions can yet be made about the activity of retinoids in in vitro test systems. which were inactive in Systems 1 and 2, an excellent correla tion was observed between activities in Systems 1 and 3 for retinyl acetate, N-(4-hydroxyphenyl)retinamide, retinylidene MATERIALS AND METHODS dimedone, W-ethylretinamide, and A/-benzoylretinylamine. Chemicals. MCA, retinyl acetate, and retinoic acid were Compounds shown to be inactive in System 1 had little or no obtained from the Sigma Chemical Co., St. Louis, Mo. Other activity in System 2. retinoids were obtained directly from Dr. M. Sporn of the However, the ability of retinoids to cause increased adhesion National Cancer Institute, who in turn obtained them from the could not be correlated with Systems 1 or 3 in all cases. For investigators listed in Chart 1, with the exception of one sample instance, retinyl acetate was highly active in Systems 1, 2, and of 13-c/s-retinoic acid obtained directly from Hoffmann-La 3, whereas retinylidene dimedone was highly active in Systems Roche Inc., Nutley, N. J. All retinoids were stored in liquid N? 1 and 3 but weakly active in System 2. Conversely, A/-(4- and freshly dissolved in dry acetone immediately prior to use. hydroxyphenyOretinylamide was highly active in Systems 1 and All solutions were handled under low-intensity "gold" fluores 3 but caused a decrease in System 2. The lack of activity of cent lighting (General Electric Co., Schenectady, N. Y.). retinoic acid isomers in the C3H/10T1/2 clone 8 system is Cells. The 10T1/2 cell line obtained from mouse embryo paradoxical and may provide important information on require fibroblasts (16, 17) was used throughout this study. Culture ments for their activation and/or transport. conditions were as previously described. Transformation Assay. The assays for transformation and INTRODUCTION cytotoxicity were performed precisely as described previously (4, 13). Cultures were exposed to a final concentration of 2.5 Retinoids, the natural and synthetic analogs of vitamin A, jug MCA per ml and 0.5% acetone, which was applied 24 hr have been demonstrated to be active in inhibiting the devel after plating. Control cultures received 0.5% acetone. Growth opment of chemically induced carcinomas at a variety of organ medium was removed from both treated and control cultures sites (for a review, see Ref. 20). Retinoids are also finding after 24 hr, and fresh prewarmed medium was added. Retinoids clinical application as inhibitors of keratinizing dermatoses and in solution in acetone were added 7 days after removal of have been reported to cause remission of squamous cell car carcinogen to give a final concentration of 0.5% acetone and cinomas and the disappearance of a number of premalignant the stated concentration of retinoid. Retinoids were readded conditions of the skin and mucous membranes (12). Because weekly after medium change. of their ability to prevent or delay the experimental induction of Cultures used to determine cytotoxicity were fixed 7 to 10 cancer in experimental animals and to cause the reversal of days after seeding, and cultures set up to determine TF's were premalignant changes in epithelium (11), the retinoids have fixed 36 days after seeding. Only type III colonies are included been called chemopreventive agents (20). in the transformation data. Approximately 90% of these can be We have recently reported that the natural derivatives of expected to be tumorigenic in immunosuppressed syngeneic vitamin A, retinyl acetate, retinol, and retinaldehyde, are highly active in inhibiting the development of MCA2-induced neoplas mice (17). The TF for individual groups was calculated as previously described (see Table 1, legend). Control cultures tic transformation in the 10TVÕ?cellline (13). In order to extend which received either acetone (0.5%) or MCA (2.5 /ig/ml) plus acetone (0.5%) utilized dishes seeded at the beginning, middle, 1Supported in part by USPHS Grant CA-25484. 2 The abbreviations used are: MCA, 3-methylcholanthrene; 10T'/2. C3H/10T'/2 and end of the experimental setup. clone 8; TF, transformation frequency; PE, plating efficiency. Adhesion Assay. Retinoids were added as described above Received November 19, 1979; accepted June 3, 1980. to cultures of 10TV? cells in 60-mm plastic Retri dishes which SEPTEMBER 1980 3141 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. J. S. Bertram COMPOUND(SOURCE) STRUCTURE U) -J -J I. Aii-trons-r.tinyiocstati w U (Sigma Ch•mscoiCo.) I.z Li Li a AIi-trons-retsøo.coc'd ö@L@@L@CcoH I (SigmaCPem'calCot a z 0z I i3-c,s-r.tinoic acid (Hotfmann- LoRoch. ae Nuilsy, Nsw JIVMy) TIME (Days) 4. p4-4- Hydroayphsnylretinomsds Chart 2. Rate of release of 101½cells from plastic substrate. Replicate ( Johnsonosid Johnson) cuftures of confluent 10T½cellswere treated at time zero with 0.5% acetone as control (x), 0.3 iag retinyl acetate per ml (Lx), or 0.03 pg retinyl acetate per ml (0). At the indicated times after treatment, cultures were washed with warm 0.9% NaCI solution and then covered with 2 ml of 0.5% trypsin:2 x iO@ N EDTA in phosphate-buffered [email protected] agitated rapidly In a warm room (37°), 5. N-Ethylr.i,nomsds ( Iloffmonn- LoRocti. and 0.2-mI aliquots were removed for cell counting as descrIbed in the text. PlutiSy,Nsw Jacisy) Results show the percentage of total cells removed after 2 mm. Points, mean of 2 counts performed on each of 2 cultures; bars, S.E. As explained In the text, retinyl acetate-treated cultures were significantly (p < 0.01) more adherent to the plastic substrate after a minimum of 3 days in culture. 6 P4-Bsozoyir.tsnyiomifl* ( BASF) were withdrawn for electronic particle counting at intervals of 1, 1.5, 2, and 4 mm after addition of the trypsin:EDTA solution. 0 At the end of this 4-mm time period, each culture was scraped 7 Rsiinyiidsol Dim.dons (P4.Actonond A.Brossi NIH) with a rubber policeman to dislodge all cells, and a 0.2-mI aliquot of this was taken for counting to give an estimate of total cells originally present in that dish. All procedures were 8. Aryl trisns onoloq of rsimo'c ocid conducted in a warm room (37°).Crudecounts were corrected (N. DowsonandP.Hobbs SRI inlsrnotionoi for coincidence and for volume changes, and the total cells MsnIo Pork, CA) dislodged from the Petri dish at each time period were calcu lated as a percentage of the total cells originally present. 9 C17—Corbosyiiconolog of Results are reported as the mean ±S.E. of the 4 replicate rsl,noic acid ( C.Hsolhcock cultures. Univ. of Coiiforn.o Bertisisy) RESULTS so Thiophsnsanalogof @cr@@22s__L.__%_.._L_..@COOH rslinOiC ocid Table 1 presents the results obtained in 2 separate experi ments. Although within a single experiment a consistent re sponse to a standard exposure to a carcinogen was obtained Chart 1. Chemical structures and sources of the retinoids used in this study. Additional addresses: BASF Aktiengesellschaft, 6700 Ludwigshafen am Rhein, [i.e., 2.24 ±0.1 transformants/dish (Table 1, Column 5, Line Germany; Johnson & Johnson, New Brunswick, N. J. 2)], quantitative differences in response were often found be tween experiments conducted weeks apart. This occurred with had reached confluence 1 to 3 days previously and were the last 2 compounds shown in Table 1, and the appropriate maintained in Eagle's basal medium supplemented with 5% controls are listed separately. heat-inactivated fetal calf serum (Grand Island Biological Co., As previously reported (13) and confirmed here (Table 1), Grand Island, N. V.). This treatment protocol was designed to retinyl acetate is a potent inhibitor of MCA-induced malignant duplicate the conditions of retinoid treatment used in the trans transformation. Of the compounds tested, only N-(4-hydroxy formation assay. For all studies except the time course studies phenyl)retinamide was of approximately equal potency, caus described in Chart 2, cultures were evaluated 4 days after ing a decrease in TF to 34% of control at a concentration of addition of retinoid. In the case of retinyl acetate, this time 0.03 @zg/mI.Atthis concentration, retinyl acetate inhibits trans period results in a maximum increase in adhesion (Chart 2).