Augmentation of 6-Aminonicotinamide Antagonism of Tumor Growth by Compounds with Estrogenic0 Activity*

L. S. DIETRICH (Department of Biochemistry, University of Miami, School of Medicine, Miami, Florida)

SUMMARY The hormonal derivatives diethylstilbestrol, hexestrol, dienestrol, cortisone, estradiol-17a, estradiol-17/3, progesterone, and phloretin were compared with regard to their capacity to enhance carcinostasis in the presence of the niacin antagonist 6- aminonicotinamide. Diethylstilbestrol and hexestrol were found to be more powerful than either estradiol-17/Ã®or dienestrol. Phloretin, estradiol-17a, and progesterone were without activity. Cortisone, either alone or in the presence of 6-aminonicotinamide, exhibited slight antitumor activity. In the absence of 6-aminonicotinamide, diethylstilbestrol, hexestrol, dienestrol, and estradiol-17/3 had no antitumor activity. The suggestive correlation between estrogenic activity and enhancement of carcino stasis in the presence of 6-aminonicotinamide is discussed.

Adenocarcinoma 755 grown in C57BL mice is sensitive materials dissolved in sesame oil were administered intra- to the niacin antagonist 6-aminonicotinamide (5). More peritoneally and intramuscularly, respectively. Adminis recently it has been shown that combined therapy of tration of the hormonal agents and vitamin antagonist diethylstilbestrol and 6-aminonicotinamide has greater was carried out on well established tumors (14-20 days antitumor effect than either drug by itself (3). When the old) and continued for 6 consecutive days. In order to activities of various cytochrome-linked pyridine nucleotide-dependent enzymes of tumors from animals treated TABLE 1 with these drugs were measured (1), it was shown that, EFFECTOF ESTROGENSONTHE GROWTHOFTHE although diethylstilbestrol had no effect on the activity ADENOCAHCINOMA755 of these enzymes, combined therapy with the hormone and cent 6-aminonicotinamide markedly increased the enzymatic Group*Sesame tumor wt, weight inhibition observed by the administration of 6-amino (gm. Â±S.E.)t1.92 change+3+4-2+ nicotinamide (6-AN). The hyperphysiological levels of the estrogens employed oilEstradiol-170HexestrolDienestrolSesameÂ±0.322.39 in these studies raised the question as to whether the tumor Â±0.302.84 Â±0.391.74 growth inhibition observed was owing to the estrogenic Â±0.311.75 1+12+5+30+ properties of the drug or to functional groups of the mole cule unrelated to hormonal action. An elucidation of this oilStilbestrolSaline Â±0.201.32 question motivated the studies to be reported. Â±0.174.53

MATERIALS AND METHODS controlCortisone Â±0.432.43 C57BL mice, 2-4 months old, weighing 18-22 gm., were acetateAverage Â±0.32ÃŽMortality0/200/200/200/200/140/150/200/20Per15 housed in plastic cages in an air-conditioned, constant- * Hormones were dissolved in sesame oil, with the exception temperature room (74Â°F.). All mice received, ad libitum, of cortisone acetate, which was suspended in saline. All hor a diet of Rockland pellets and water. The neoplasm em mones were given at a level of 10 mg/kg for 6 consecutive days. ployed was the mammary Adenocarcinoma 755, trans t S.E. = standard error = planted into the axillary region by the usual trocar technic. n- 1) 6-Aminonicotinamide, dissolved in saline, and estrogenic ÃŽ"t" value of this experiment was 2.7. * This work was supported in part by Grant CY-4868 and a The results of two additional experiments with each of the Career Development Award GM-K3-15,186 from the United States estrogens were similar to the tabulated examples. In three Public Health Service. other experiments with cortisone acetate no inhibition was Received for publication July 5, 1963. obtained. 61

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1964 American Association for Cancer Research. 02 Cancer Research Vol. 24, January 1964 note any delayed host toxicity, a waiting period of 5-6 The effect of combining 6-aminonicotinamide and vari days of no treatment was observed after the last injection. ous estrogenic and nonestrogenic substances is presented At the end of this period the animals were sacrificed, the in Tables 2 and 3. Diethylstilbestrol, at a level of 1 mg/ tumors removed, and wet weights were determined to the kg, had no effect on tumor growth when given along with nearest 10 mg. the niacin antagonist 6-aminonicotinamide. Increasing the level of estrogen to 4 mg/kg resulted in a significant RESULTS suppression of tumor growth as compared with 6-AN The effect of the various hormone derivatives on the alone. Raising the level of hormone administered to 10 growth of the 755 tumor is presented in Table 1. The mg/kg produced no greater tumor toxicity but seemed to compounds hexestrol, diethylstilbestrol, dienestrol, and increase host toxicity. In the case of hexestrol and estradiol-17/3, when administered at a level of 10 mg/kg, dienestrol neither compound exhibited activity when ad did not inhibit the growth of the 755 tumor and exhibited ministered at 1 mg/kg. Raising the level of hexestrol to little host toxicity. Cortisone acetate, although exhibit 4 mg/kg resulted, in a majority of the experiments, in a ing no apparent host toxicity, significantly inhibited significant suppression of tumor growth, as compared tumor growth in one experiment out of four. with the effect of 6-AN alone. No effect was observed at

TABLE 2 EFFECT OFCOMBININGO-AMINONICOTINAMIDEANDVARIOUSESTROGENICANDNONESTROGENICSUBSTANCES ON THE GROWTHOF ADENOCARCINOMA755

Level of cent estrogen tumorwt.(gm. weight (mg/kg)1410141014101010Exp.no.123123t12345Group6-AN Â±S.E.)2.41 change-3+4+3+2+

oil6-AN+ ses. Â±0.272.34 +stilbestrol6-AN Â±0.243.01

oil6-AN+ ses. Â±0.281.84 +stilbestrol6-AN Â±0.25*3.26

oil6-AN+ ses. Â±0.41*1.50 19+ +stilbestrol6-AN Â±0.252.41 10+3-1+6+2+2+5+

oil6-AN+ ses. Â±0.271.65 +hexestrol6-AN Â±0.222.12 +dienestrol6-AN Â±0.313.01

oil6-AN+ ses. Â±0.281.99 +hexestrol6-AN Â±0.25*2.26 +dienestrol6-AN Â±0.233.26

oil6-AN+ ses. Â±0.411.46 19+ + hexestrol Â±0.24* 12 6-AN +dienestrol6-AN 1.63 Â±0.34*2.41 9/210/173/181/182/191/207/190/181/200/200/200/201/20Per+13-3+1+3+4+

oil6-AN+ ses. Â±0.272.27 -17/36-AN+ estradici Â±0.333.01

oil6-AN+ ses. Â±0.282.43 +estradiol-17/36-AN Â±0.243.26

oil6-AN-f ses. Â±0.41*1.00 19+10+11+9-4-4+7+4 +estradiol-17/36-AN Â±0.231.18

oil6-AN+ ses. Â±0.181.22 +estradiol-17a6-AN Â±0.111.43 +progesterone6-AN Â±0.140.74 +cortisone6-AN Â±0.121.30

oil6-AN-f ses. Â±0.181.49 + phloretinAv. Â±0.21Mortality0/170/171/183/191/202/200/171/182/181/182/193/181/203/20 6-Aminonicotinamide dissolved in saline was administered at a level of 2 mg/kg. The hormonal derivatives, given at a level of 10 mg/kg, were dissolved in sesame oil with the exception of cortisone acetate, which was suspended in saline. Therapy was begun 14 days after transplantation and con tinued for six consecutive injections. * Experiments exhibited "t" value of 2.6 or higher.

TABLE 3 administered none of the estrogenic substances by them SUMMARYOF BIOLOGICALSTUDIES CONCERNINGTHE EFFECT OF selves demonstrated either tumor or host toxicity (Table STEROIDDERIVATIVESONCARCINOSTASISINTHE PRESENCEOF 1). However, when the estrogenically active derivatives 6-AMINONICOTINAMIDE were combined with nontoxic levels of 6-AN, increased

No. EXP. HAVING "I" VALUES host and tumor toxicity was observed. The host toxicity OF 2.6 OR HIGHER was particularly noticeable in the case of diethylstil COMPOUNDStilbestrolDienestrolHexestrolEstradiol-17/3PhloretinProgesteroneCortisoneEstradiol-17aTOTALEXP.10 bestrol and hexestrol. In these studies, however, there was no consistency between host toxicity and inhibition mg/kg6/113/53/53/50/30/41/40/44mg/kg4/60/33/30/31mg/kg0/60/30/30/3of tumor growthâ€”i.e., marked host toxicity was observed in experiments showing no tumor toxicity, and significant antagonism of tumor growth occurred in experiments showing little host toxicity. Thus, it is doubtful whether host toxicity per se played a significant role in retarding tumor growth in these cases. The action of cortisone in these studies is not under stood. When cortisone was used, significant antagonism of tumor growth was observed in one experiment out of mi â€”m* nil â€”treatment with 6-AN. four in the case of hormone therapy alone (Table 1) and "Â¡2=treatment with Estrogen + one experiment out of four in the presence of hormone and + 6-AN. 6-AN (Table 3). The other compounds tested were The results are considered consistent beyond chance variation when "/" is 2.6 or higher. Adenocarcinoma 755 was grown in totally inactive in the absence of 6-AN. C57BL mice. Therapy was begun on well established tumors Whether the effect of these compounds on neoplastic (14-21 days after transplantation). The compounds were given growth is a direct or indirect action of the estrogenic at the dosage indicated for 6 consecutive days. activity of these substances is not known. However, the TOIand mi refer to mean tumor weights of groups of at least extremely high levels of estrogen necessary to elicit a seventeen animals. response and the lack of knowledge whether the activity observed is directly or indirectly of estrogenic nature this level of dienestrol. Increasing the level of hexestrol should not distract from the fact that this experimental to 10 mg/kg resulted in no apparent increase in toxicity. neoplasm appears to exhibit hormonal sensitivity. Fur In the case of dienestrol, however, increasing the level thermore, this suggestive hormone sensitivity is appar administered to 10 mg/kg resulted, in the majority of ently in some manner linked to pyridine nucleotide experiments, in a significant inhibition of tumor growth metabolism, since, in the case of diethylstilbestrol, this as compared with that from 6-AN by itself. compound has been shown to enhance 6-AN antagonism Estradiol-17(3 exhibited antitumor activity, in the pres of various pyridine nucleotide-dependent enzymatic sys ence of 6-AN, to the same degree as dienestrol â€”i.e.,it was tems (1). active only at the 10 mg/kg level. The nonestrogenic ACKNOWLEDGMENTS isomer, estradici -17Â«,wasinactive at a level of 10 mg/kg. The author wishes to acknowledge the technical assistance of Progesterone and phloretin at a level of 10 mg/kg com Miss Peggy Hayworth. bined with 6-AN were inactive. Cortisone exhibited slight activity â€”i.e., in one experiment of four significant REFERENCES 1. DIETRICH, L. S., ANDMARTIN, D. S. Augmentation of 6-Amino- inhibition of tumor growth occurred when the 6-AN and nicotinamide Antagonism of DPN-dependent Enzymatic cortisone groups were compared with the 6-AN. It Systems by Diethylstilbestrol. Cancer Res., 21:361-64, 1961. should be pointed out, however, that cortisone by itself 2. DOBBS, E. C., ANDLAWSON,W. Molecular Structure in Rela showed slight antitumor activity. tion to Estrogen Activity: Compounds without a Phenan- threne Nucleus. Proc. Roy. Soo. London, s.B, 125:222-32, DISCUSSION 1938. 3. MARTIN, D. S.; DIETRICH, L. S.; AND FUGMANN, R. R. Hor The compounds studied exhibited estrogenic activity monal Augmentation of Antimetabolic Chemotherapy. Proc. in the following approximate order of decreasing activity: Soc. Exp. Biol. Med., 103:58-60, 1960. hexestrol, diethylstilbestrol, estradiol-17/3, dienestrol, 4. SHAPIRO, D. M. Combination Chemotherapy with 8-Azagua- nine and Sex Hormones on a Mouse Mammary Carcinoma. and phloretin (2, 6). Phloretin is an extremely weak Cancer Res., 12:713-15, 1952. estrogen (2). The other compounds are without estro 5. SHAPIRO,D. M.; DIETRICH, L. S.; ANDSHILS, M. E. Quantita genic activity. The capacity of these compounds to en tive Biochemical Differences between Tumor and Host as a hance the carcinostasis of 6-AN appeared to be as follows: Basis for Cancer Chemotherapy. V. Niacin and 6-Aminonico- hexestrol and diethylstilbestrol are the most active, fol tinamide. Cancer Res., 17:600-604, 1957. 6. SOLMSSEN,U. V. Synthetic Estrogens and the Relation between lowed by estradiol-170 and dienestrol. The compounds Their Structure and Their Activity. Chem. Rev., 37:481-598, phloretin, progesterone, and estradici- 17Â«were without 1945. activity. Thus, there is a suggestive correlation between estrogenic potency between hexestrol, diethylstilbestrol, and the capacity of these compounds to enhance carcinostasis estradiol-17/3 to permit correlation with tumor-inhibitory activity. in the presence of the niacin antagonist 6-AN and the This should not, however, distract from the observations that estrogenic activity of the substance in spite of the massive compounds lacking estrogenic activity, with the possible excep levels of estrogen administered (10 mg/kg).1 At the levels tion of cortisone, did not inhibit tumor growth in the presence of 6-ANâ€”the most noteworthy comparisons being estradiol-17/3, 1 One might argue that there are insufficient differences in which is active, and estradiol-17a, which is inactive.

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1964 American Association for Cancer Research. Augmentation of 6-Aminonicotinamide Antagonism of Tumor Growth by Compounds with Estrogenic Activity

L. S. Dietrich

Cancer Res 1964;24:61-63.

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