Structure-Activity Relationships Among Various Retinoids and Their

[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 10TVÃ•?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

COMPOUND(SOURCE) STRUCTURE

U) -J -J I. Aii-trons-r.tinyiocstati w U (Sigma Châ€¢mscoiCo.) I.z Li Li a AIi-trons-retsÃ¸o.coc'd Ã¶@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 101Â½cells from plastic substrate. Replicate ( Johnsonosid Johnson) cuftures of confluent 10TÂ½cellswere 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 (37Â°), 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 (37Â°).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 C17â€”Corbosyiiconolog of Results are reported as the mean Â±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 Â±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). formation by 50%. Both compounds cause no detectable cy Four replicate cultures for each concentration of retinoid and totoxicity at concentrations inhibitory to transformation. The for each time point were washed briefly with 0.9% NaCIsolution dimedone analog exhibited high activity but was marginally and then covered with 2 ml of 0.5% trypsin (1:250; Grand less potent on a weight basis than was retinyl acetate or N-(4- Island Biological Co.):2 x i0@ M disodium EDTA (Eastman hydroxyphenyl)retinamide. Both synthetic compounds and Kodak Co., Rochester, N. V.) in phosphate-buffered saline retinyl acetate were of equivalent toxicity. Of the other com (0.8% NaCI:0.1 15% Na2HPO4:0.02% KH2PO4.2H2O:0.02% pounds tested, only the aryltriene analog decreased transfor KCI, pH 7.4) at 37Â°.Culture dishes were then vibrated on an mation frequency without inducing major toxicity. However, at orbital shaker (BelIco, Vineland, N. J.), and 0.2-mI samples the high concentration of 1.0 pg/mI required to produce an

3142 CANCER RESEARCH VOL. 40

Table 1 Effects of various retinoids on PE and on MCA-induced TF in the 10T/? cell line To determine PE, cells were continuously exposed to the retinoid, which was added 24 hr after plating. To determine effects on transformation, retinoids were added to cultures previously initiated with MCA as described in the text. con centration (% of con of (% of con Treatment8Acetone (/ig/ml)000.30.10.030.013.01.00.30.10.033.01.00.30.10.030.013.01.00.30.13.0100.30.10.30.10.030.010.0030.30.10.030.010.0033.01.00.301.00.30.10.033.01.00.30.1PEtrol)100(25)1009610791952.7677996991.55284979699284382766880717030405274933238671029791101116100(25)94961019810110797108No.dishes"35361111911ND'ND101212ND121212121212121212ND12121210121212NDND118111012121112121111121212ND12Transformants/dish002.24 trol)01004.218.754.681.011.215.333.900268120.090.6043.159.1113.151.3120.084.30147.7144.2105.1171.6133.187.5100.469458.991.510016.288.158.795.1123.1131.5146.3 (0.5%)MCAcontrol Â±0.1Â°0.090.45 /Â¿g/ml)Retinylcontrol (2.5

acetateA/-<4-Hydroxyphenyl)retin-amideRetinylidene Â±0.21.11 Â±0.21 .73 Â±0.30.2

Â±0.10.33 Â±0.10.75 Â±0.3000.58

dimedoneN-EthylretinamideN-BenzoylretinylamineAll-frans-retinoic

Â±0.32.58 Â±0.42.0 Â±0.300.42

Â±0.11.08 Â±0.21.92 Â±0.20.92

Â±0.31.92 Â±0.21.33 Â±0.401.33 acid91 Â±0.41.67 Â±0.31.75 Â±0.31.45

acid9Thiophene3-cjs-Retinoic Â±0.22.0 Â±0.32.0 Â±0.22.2 Â±0.21.42

analogMCA Â±0.31.33 Â±0.42.05 Â±0.21 fig/ml)"Aryltrienecontrol (2.5 .08 Â±0.30.17

analogCi? Â±0.10.91 Â±0.40.64 Â±0.31.0 Â±0.21

carboxylic acid analogRetinoid .33 Â±0.41.42 Â±0.31.58

Â±0.3TP"<0.010.900.040.170.490.730.10.130.3000.241.10.8100.380.521.010461.0707501.321.290941.541.190.780.90.620.530.820.430.070.380.250.410.530.560.63TF a For structures of retinoids. see Chart 1; for biological activity in other systems, see Table 2. For evaluation in the transformation assay. '' Type III morphological transformants. " Calculated as:

No. of transformants/dish X 100 No. of surviving colonies/dish To obtain the number of surviving colonies/dish, multiply PE control by 2.5 tor a control PE of 25%. " Mean Â±S.E. ' ND, not determined. 9 Representative results from 3 separate experiments. *"Carcinogen-only control for the aryltriene and Ci? carboxylic acid analogs.

SEPItMbtR 1900 3143

effect with this compound, the colonies in the cultures plated 3.0 @sg/ml. On the confrary, a consistant but statistically insig to determine PE were smaller than controls and the cells nificant increase in TF was observed at all concentrations appeared flattened. Both N-ethylretinamide and N-benzoylre tested. The thiophene analog induced marginal toxicity at a tinylamine caused toxicity over all concentration ranges tested high concentration of 3.0 @g/mlandcaused a small decrease and at concentrations of 1 @g/mlinhibitedtransformation only in TF at this concentration. At 1.0 @g/ml,transformation was by about 50%, making both compounds about 30-fold less depressed to about 60% of control without causing toxicity. potent than retinyl acetate, N-(4-hydroxyphenyl)retinam This analog thus appears to have marginal activity in the 10T14 ide, and the dimedone analog. assay system. Of great surprise was the finding that all-trans-retinoic acid Effects of Retinolds on Adhesion. For time course studies, and its 13-cis isomer were both inactive in causing a decrease recently confluent cultures of 10TÂ½cells were treated with in MCA-induced transformation frequency, although both com 0.03 or 0.3 @tg/mlretinyl acetate in acetone or 0.5% acetone pounds were highly toxic as measured in the PE assay. The TF alone as control. Analysis of replicate cultures for increased calculated for these compounds may be artifactually high be adhesiveness to the substrate at increasing times after treat cause of the cytotoxicity measured in the dishes seeded for TF ment showed a similarity between treated and control cells determination. Since conditions of treatment with retinoids when analyzed immediately after treatment, but, whereas a were not identical for these 2 assays (for PE, cells are treated decrease in adhesion was seen in control cells 1 day after 1 day after plating so that the effects on single cells may be treatment, retinoid-treated cells remained more firmly attached determined, whereas, for TF, cells are treated 9 days after to the plastic Petri dish. After 3 days treatment, the difference seeding to eliminate effects of retinoids on the initial events in between treated and control cultures was statistically signifi carcinogenesis), it may not be valid to assume that equal cant and remained so throughout the duration of the experi cytotoxicity will occur under both conditions. However, from ment (Chart 2). The data shown in Chart 2 are for cells released Table 1 it will be seen that toxic concentrations of retinoic acid after a 2-mm exposure to trypsin:EDTA, a time period sufficient or 13-cis-retinoic acid do not strongly inhibit transformation to cause maximum release of cells by this method (see Charts measured on the basis of transformants/dish and that signifi 3 and 4). Because of this time delay between treatment and cant inhibition only occurred at the highest, most toxic, con response, the effects of the various synthetic retinoids were centrations tested. The experiments with all-trans-retinoic acid examined after 4 days of treatment. and 13-cis-retinoic acid were conducted on 3 separate occa Effects of Various Retinolds on Cell Adhesion. In Charts 3 sions with different batches of drug; the results on all occasions and 4 are presented data to show the time course of release of were quantitatively similar to those reported in Table 1. cells into a trypsin:EDTA solution from plastic Petri dishes. Two compounds, the C17 carboxylic acid analog and the Replicate cultues were analyzed 4 days after receiving the thiophene analog of retinoic acid, which have been reported to appropriate acetone or retinoid treatment. As seen in Chart 3A, be inactive in other test systems (Chart I ), were examined in approximately 15% of the cells were released after 1 mm of the 10TÂ½cellline as negative controls. The C17carboxylic acid exposure and a plateau was reached after 2 mm of exposure. analog, which differs from retinoic acid in being 3 carbons Moreprolongedexposurethan4 mmtotrypsin:EDTAresulted short in the side chain, was inactive in causing toxicity or in in an apparent decrease in release of cells, as measured by reducing MCA-induced transformation at concentrations up to electronic particle counting, that was caused by the irreversible clumping of cells (data not shown). To avoid this, we added 10% fetal calf serum at this time, which prevented clumping

20 (@ 10 k@=::#@TE

2 30

10 I C D @ I 2 4 2 3 4 TIME(mm)

Chart 3. Effects of retinolds active in the transformation assay on the rate of I 2 3 4) 2 3 release of trypsin:EDTA-treated cells from a plastic substrate. 10TÂ½cellswere TIME (mm) grown to confluence and 4 days prior to assay treated with the appropriate retinoid or acetone as control. The rate of release of cells induced by a trypsin: Chart 4. Effects of retinolds inactive or weakly active In the transformation EDTA solution was determined in 4 replIcate cultures as described in â€œMaterials assay on the rate of release of trypsin:EDTA-treated cells from a plastic substrate. and Methods.â€•x , low dose of the respective retinoid; 0, hIgh dose of the Experimental protocol as in Chart 3. x , low dose of the respective retinold; 0, respective retinold. A, 0.3 and 0.03 @ogretinylacetate per ml and 0.5% acetone high dose of the respective retinold; controls as In Chart 3A. A, 0.3 and 0.1 @g control(i@).B, 0.3 and 0.1 @gretinylidenedimedone per ml. C, 0.3 and 0.1 @gN- retinoic acid per ml. B, 0.3 and 0. 1 @ig13-cis-retlnoic acid per ml. C, 3.0 and 1.0 (4-hydroxyphenyl)retinamide per ml. 0, 3.0 and 1.0 gigN-ethylretinamlde per ml. gogC,7carboxylic acid analog per ml. D, 3.0 and 0.3 pg thlophene analog per ml.

3144 CANCERRESEARCHVOL. 40

and allowed us to obtain a total cell count as described in MCA-induced transformation to 43 and 0% of controls, respec â€œMaterialsandMethodsâ€•.Manipulation of trypsin and/or tively (Table 1). EDTA levels was not successfulin overcomingthis problem, Retinoids shown to be essentially inactive in the transfor and we were not able to achievea morecompletedetachment mation assay (i.e., all-trans-retinoic acid, 13-cis-retinoic acid, of cells from the substrate without scraping. As seen in Chart and the C17carboxylic analog of retinoic acid) also exhibited 3A, retmnylacetate caused a highly significant (p < 0.01) little activity in the adhesion assay (Chart 4, A, B, and C). Even decrease in the rate at which cells were releasedfrom the at a concentration of 0.3 @gofretinoic acid per ml, which was substrate, and a clear dose-response relationship was ob highly toxic and which eliminated the induction of transformed served. foci in the assay system, this compound caused only a weak The dimedone analog, which at the concentrations tested increase in cellular adhesion (Chart 4A). This increase became (0.1 and 0.3 @g/ml)inhibited transformation to 26 and 0% of significantly different from controls when data points from both controls, respectively (Table 1), caused a significant (p < 0.01) concentrations and from time points between 1.5 and 4 mm, increasein adhesiononly at the 0.3 @g/mllevel(Chart 3B). inclusive, were combined. The thiophene analog, which was Conversely, N-(4-hydroxyphenyl)retinamide at concentrations weakly active in the transformation assay (Table 1), caused a (0.1 and O.3@tg/ml), which caused little toxicity but pronounced significantly different (p < 0.05) increase in adhesion at the inhibition of transformation (Table 1), caused a significant highest concentration tested of 3.0 @g/ml. decrease(p < 0.01) in adhesionof 10TÂ½cellsto plastic. It should be noted that the lower concentration caused the more DISCUSSION pronounced response (Chart 3C). Similarly, N-ethylretinamide, the only other retinamide tested (Chart 3D), also caused a The results presented in Table 1 and summarized in Table 2 significant (p < 0.01 ) decrease in adhesion of cells at the lower show that inhibition of carcinogen-induced neoplastic transfor concentration tested (1.0 pg/mI) but had no effect on adhesion mation in the 10TÂ½cell culture system provides a good mdi at the higher concentration of 3.0 @g/ml.Theseconcentrations cation of the chemopreventive activity of several classes of producedpronouncedcytotoxicityin 1OT'I4cellsand reduced natural and synthetic retinoids but that all-trans-retinoic acid

Table 2 retinoidsCompoundaBiologicalStructure-activity relationships between invivo and in vitro activities of activityIn

vitroGrowth vivoIn

trachea of pro of (50% effective Cytot@x- transforma adhe sion@'AII-trans-retinyl motionInhibitioncarcinogenesisHamsterdose) icityInhibition tion@'Increased +N-(4-: acetate+ + +c (2)+ + +d.@. .@. ,@, ++ + 10-Â°Me.@

+â€”Retinylidene;jdroxyphenyl)retinamide+ (15)+ + 4 (15)+ + + (1 5) Â± + 3 x 10'Â°M+

dimedone+eND+ + + Â± + + +++(7)Â±N-Ethylretinamide+e+ 2xlOb0Me+

(1)N-Benzoylretinylamlne+e+ + + (22)+ + + += 2 x 10Â°M+

+@+ + + 10Â°MÂ°+NDAll-trans-retinoic 4 x

10)1 acid+ + + (23)+ +d (5)+ + (1 9) + + + + + (1, 3 x 10'Â° N 8 x 10â€• Meâ€”Â±

@ .@. ,@. ,@, 10)Aryltriene3-cis-Retinoicacid+ + + (24)+ + 4.(9).@. + + Ã· (1 , 10-10 MeÂ±

analog+eND+ + + 2x1O@0Me+4NDThlophene

analogNDNDâ€” â€” lO.8Me++C17 >1 x

CarboxylicacidanalogNDNDâ€” â€” >1 x 1O@8Meâ€”â€” a For structures and sources, see Chart 1. b Results from this paper, except where indicated otherwise. C ND, not determined. d Active, but with marked toxicity. . personal communication. M. B. Spom.

SEPTEMBER1980 3145

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. J. S. Bertram and its isomer, 13-cis-retinoic acid, are not active in this adhesion in cultured transformed mouse fibroblasts. J. NatI. Cancer Inst., system. This finding is perplexing, since in in vivo and in several 62: 1473-1478, 1979. 2. Ames, S. R. Bioassay of vitamin A compounds. Fed. Proc., 24: 917â€”923, in vitro assays these compounds possess among the highest 1979. activities in inhibiting carcinogen-induced neoplasia or in main 3. Arens, J. F., and Van Dorp, D. A. Synthesis of some compounds possessing vitamin A activity. Nature (Lond.), 157: 190â€”191, I 946. taming normal epithelial differentiation (Table 2). In addition to 4. Bertram, J. S. Effects of serum concentration on the expression of carcino the lack of activity of these 2 retinoic acids in the transformation gen-induced transformation in the C3H/ 101Â½CL8 cell line. Cancer Res., system, neither of these compounds produced major effects 37: 514-523, 1977. 5. Bollag, W. Prophylaxis of chemically induced benl9n and malignant epithellal on cell adhesion to a plastic substrate such as was seen with tumors by vitamin A acid (retinoic acid). Eur. J. Cancer, 8: 689â€”693,1972. retmnylacetate(Chart 3A). This finding is again surprising, since 6. Chytil, F., and Ong, D. E. Cellular retinol and retinoic acid binding proteins Adamo et a!. (1) and Jetten et a!. (10) have described very in vitamin A action. Fed. Proc., 38: 2510â€”2514,1979. 7. Dickens, M. S., Custer, R. P., and Sorof, S. Retlnoid prevents mammary pronounced effects of similar concentrations of retinoic acid in gland transformation by carcinogenic hydrocarbon in whole-organ culture. causing increased adhesion of BALB/3T1 2 cells. Proc. NatI. Acad. Sci. U. S. A., 76: 5891-5895, 1979. 8. Embleton, M. J., and Heidleberger, C. Neoantigens on chemically trans The reason for this lack of activity of retinoic acids in causing formed cloned C3H mouse embryo cells. Cancer Res., 35: 2049â€”2055, effects other than toxicity in 10TÂ½cells is puzzling. Failure 1975. could be due to lack of specific binding proteins (6), inability to 9. Grubbs, C. J., Moon, R. C., Squire, R. A., Farrow, G. M., Stinson, S. F., Goodman, D. G., Brown, C. C., and Sporn, M. B. 13-cis-Retinolc acid: have access to critical intracellular sites, or failure to be met inhibition of bladder carcinogenesis Induced In rats by N-butyl-N-(4-hydrox abolically activated, although recent studies suggest that me ybutyl) nitrosamine. Science (Wash. D. C.), 198: 743-744, 1977. tabolism is not required (18). Finally, inherent deficiencies in 10. Jetten, A. M., Jetten, M. E. A., Shapiro, S. S., and Poon, J. P. Characterl zation of the action of retinoids on mouse fibroblast cell lines. Exp. Cell Res., the 10T1/2line must be considered, although this too seems 119: 289-299, 1979. unlikely in view of the past success of this line in reproducing 11. Lasnitzki, I. Reversal of methylcholanthrene-induced changes in mouse prostates in vitro by retinoic acid and its analogues. Br. J. Cancer, 34: 239â€” in vivo responses (8, 13, 14, 16). 248, 1976. From the data presented in Chart 3, it may be concluded that 12. Mayer, H., BoIlag, W., HÃ¤nni,R.,and RÃ¼egg,A.Retinolds, a new class of retinoid-induced increases in cellular adhesion do not correlate compounds with prophylactic and therapeutic activities in oncology and dermatology. Experlentia, 34: 1105-1 118, 1978. with their inhibitory effects on neoplastic transformation. Thus, 13. Merriman, R. L., and Bertram, J. S. Reversible inhibition by retinoids of 3- while retinyl acetate induced highly significant dose-dependent methylcholanthrene-induced neoplastic transformation in C3H/ 10TÂ½clone increases in cellular adhesion (Chart 3A), N-(4-hydroxy 8 cells. Cancer Res., 39: 1661â€”1666,1979. 14. Mondal, S., and Heidelberger, C. Transformation of C3H/1 0TÂ½CL8mouse phenyl)retmnamide,which was as active as retinyl acetate in embryo fibroblasts by ultraviolet irradiation and a phorbol ester. Nature inhibiting transformation (Table 1), caused a reduction of cel (Lond.),260:710â€”711,1976. lular adhesion at the lowest concentration tested and had no 15. Moon, A. C., Thompson, H. J., Becci, P. J., Grubbs, C. J., Gander, R. J., Newton, D. L., Smith, J. M., Phillips, S. L., Henderson, W. R., Mullen, L. T., effect at the highest concentration used (Chart 3C). Similar Brown, C. C., and Sporn, M. B. N-(4-Hydroxyphenyl)retinamide, a new results were obtained with N-ethylretmnamide,the other retin retinoid for prevention of breast cancer In the rat. Cancer Res., 39: 1339â€” 1346. 1979. amide tested (Chart 3D). It is of interest that Adamo et a!. (1) 16. Reznikoff, C. A., Bertram, J. 5., Brankow, D. W., and Heidelberger, C. also reported that amide derivatives of retinoic acid did not Quantitative and qualitative studies on chemical transformation of cloned cause increased adhesion of 3T12 cells. The inverse dose C3H mouse embryo cells sensitive to postconfluence Inhibition of cell division. Cancer Res., 33: 3239-3249, 1973. responses observed in Chart 3C and D, suggest that retinam 17. Reznikoff, C. A., Brankow, D. W., and Heidelberger, C. Establishment and ides may have several competing effects on target cells. The 2 characterization of a cloned line of C3H mouse embryo cells sensitive to compounds included as negative controls, namely, the C17 postconfluence Inhibition of cell division. Cancer Res., 33: 3231â€”3238, 1973. carboxylic acid analog, which has a shorter side chain than 18. Roberts, A. B., and Frolik, C. A. Recent advances in the in vivo and in vitro retinoic acid, and the thiophene analog, did not reduce MCA metabolism of retinoic acid. Fed. Proc., 38: 2524â€”2527,1979. 19. Spom, M. B., Dunlop, N. M., Newton, D. L., and Henderson, W. R. Relation induced neoplastic transformation and had no major effects on ships between structure and activIty of retinoids. Nature (Lond.), 263: 110â€” cellular adhesion. 113, 1976. Further work is in progress to determine reasons for the 20. Sporn, M. B., Dunlop, N. M., Newton, D. L., and Smith, J. M. Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids). inactivity of retinoic acids in the 10T1I4cell line. Fed. Proc.,35:1332-1338, 1976. 21. Spom, M. B., Newton, D. L., Smith, J. M., Acton, N., Jackson, A. E., and Brossi, A. Retinoids and cancer prevention: the Importance of the terminal ACKNOWLEDGMENTS group of the retinoid molecule in modifying activity and toxicity. In: A. C. Griffin and C. R. Shaw (eds.), Carcinogens: Identification and Mechanism of The excellent technical assistance of Jo Ellen Budnick, Linda Bergen, Dona Action, pp. 441 -453 New York: Raven Press, 1979. Alleyne, and 5ashi Fuji Is greatly appreciated. I also wish to thank Dr. Michael 22. Thompson, H. J., and Becci, P. J. Inhibition of urinary bladder cancer in the Sporn and Dianne Newton for help in obtaining many of the compounds listed In rat by retinoids. Proc. Am. Assoc. Cancer Res., 20: 96, 1979. Chart 1 and for providing unpublished data on their biological actMtles. 23. Zile, M. H., and DeLuca, H. F. Retinoic acidâ€”some aspects of growth promoting activity in the albino rat. J. Nutr., 94: 302â€”308,1968. REFERENCES 24. Zile, M. H., Emerick, R. J., and DeLuca, H. F. Identification of 13-cis-retinoic acid in tissue extracts and its biological activity in rats. Blochim. Biophys. 1. Adamo, S.. DeLuca, L. M., Akalovsky, I., and Bhat, P. V. Retinoid-induced Acta,141:639-641,1967.

3146 CANCERRESEARCHVOL. 40

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. Structure-Activity Relationships among Various Retinoids and Their Ability to Inhibit Neoplastic Transformation and to Increase Cell Adhesion in the C3H/10T½ CL8 Cell Line

John S. Bertram

Cancer Res 1980;40:3141-3146.

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