Effects of Folinic Acid on Amethopterin-induced Inhibition of the Ehrlich Ascites Carcinoma ALAN C. SARTORELLI,*HERBERTF. UPCHURCH,ANDBARBARAA. BOOTH* (fiiomedical Division of The Samuel Roberts Noble Foundation, Inc., Ardmore, Oklahoma) SUMMARY The effects of folinic acid on amethopterin-induced inhibition of growth, purine nucleotide biosynthesis, and DNA thymine formation were measured in Ehrlich ascites cells in vivo.Folinic acid prevented the amethopterin-induced prolongation of survival time of tumor-bearing mice at folinic acid levels which caused complete reversal of the inhibition of glycine-2-C14 incorporation into purines by the folie acid antagonist. In contrast, the depression in the rate of conversion of orotic acid-6-C14 to DNA thymine by amethopterin was only partially spared by folinic acid. The prevention of ameth- opterin's inhibitory effects by folinic acid did not appear to be due to prevention of the uptake of drug by ascites cells. The findings are discussed with respect to mechanism of action of the folie acid antagonists. Folinic acid1 has been shown to prevent the relationship. Schrecker et al. (21) recently showed toxicity of folie acid antagonists in rodents (2, 7, that large doses of folinic acid prevented the inhi 10, 17) and to reverse the antineoplastic properties bition of purine biosynthesis by folie acid antago of this class of antimetabolites (4, 8, 9, 16). nists in leukemic spleens of L1210-bearing mice. Furthermore, the growth inhibition caused by Since the biosynthesis of DNA thymine is also amethopterin in cultured mammalian cell lines markedly inhibited by folie acid antagonists, it was found to be competitively reversed by folinic was of interest to compare the effects of folinic acid (1, 11). Since folinic acid is a tetrahydrofolic acid on the amethopterin-induced inhibition of acid derivative, this appeared to suggest that a the biosynthesis of polynucleotide purines and reaction(s) in addition to the reduction of folie DNA thymine. The results indicated that com acid is inhibited by the folie acid antagonists. plete prevention of the inhibition of purine forma Amethopterin-induced inhibition of formate-C14 tion was obtained at levels of folinic acid which incorporation into nucleic acid purines was shown only partially prevented the drug-induced inhibi by Skipper et al. (22) to be partially reversed by tion of DNA thymine synthesis. folie acid. In rabbit bone marrow, folinic acid partially prevented the reduction of formate-C14 MATERIALS AND METHODS incorporation into DNA brought about by aminop- Experiments were performed on 6- to 11-week- terin, whereas folie acid was ineffective (23). old female Ha/ICR Swiss mice purchased from Winzler et al. (25), employing suspensions of leu- the A. R. Schmidt Co., Madison, Wis. Tumor kemic leukocytes, found that relatively high con transplantation was carried out by aseptically centrations of folinic acid were more effective than withdrawing ascites fluid from a donor mouse was folie acid in reversing the inhibition of formate bearing a 7-day Ehrlich ascites tumor growth. incorporation into these cells by amethopterin. The fluid was centrifuged for 2 minutes in a clinical The manner of reversal suggested a competitive centrifuge (1600 X g), supernatant peritoneal fluid was decanted, a 10 X dilution with isotonic saline * Present address: Department of Pharmacology, Yale was made, and 0.1 ml. (approximately 2 X IO6 University School of Medicine, New Haven, Connecticut. 1The following abbreviations are used: folinic acid, N5- ascites cells) of the cell suspension was inoculated formyltetrahydrofolic acid; DNA, deoxyribonucleic acid; NA, intraperitoneally into each animal. In all experi nucleic acid; AS, acid-soluble. ments, mice were maintained on Rockland rat Received for publication August 11, 1961. chow pellets and water ad libitum. 102 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. SARTORELLIetal.—Effectsof Folinic Acid 103 Survival experiments.—Mice were distributed tion of survival time of mice bearing the Ehrlich into groups of comparable weight, and therapy ascites carcinoma. Folinic acid by itself did not was begun 24 hours after tumor implantation. affect survival; however, concentrations of folinic Treatments were continued once daily for 6 con acid from 1.25-10 ing/kg body weight completely secutive days. Drugs were administered in vol prevented the tumor inhibition produced by 0.5 umes of 0.25-0.5 ml. In each experiment, tumor- mg/kg of amethopterin. bearing animals receiving injections of a compa The effects of folinic acid and amethopterin on rable volume of vehicle were included to serve as purine biosynthesis de novo were measured, and controls. Animals were weighed during treatment, the results obtained are shown in Table 2. Gly- and excessive weight loss was used as an indica cine-2-C14 incorporation into polynucleotide and tion of drug toxicity. The survival time of groups acid-soluble adenine was used as the measure of of tumor-bearing animals was used as the criterion purine formation. Folinic acid at 5 mg/kg had no of tumor inhibition. effect on purine synthesis but at 10 mg/kg caused Biocliemical experiments.—Amethopterin and a slight stimulation of glycine incorporation. folinic acid (Lederle Div., American Cyanamid Amethopterin treatment produced approximately Co., New York, N. Y.) were dissolved in isotonic 75 per cent retardation of uptake of isotopie pre sodium bicarbonate and injected intraperitoneally cursor. Increasing doses of folinic acid caused a into mice bearing a 6-day ascitic growth. At continuous lessening of amethopterin-induced in various times after these injections, each mouse hibition: at a folinic acid concentration of 2.5 mg/ received an intraperitoneal injection either of 100 kg, amethopterin produced 35 per cent inhibition; fig. of glycine-2-C14 (Tracerlab, Inc., Waltham, at 5 mg/kg of folinic acid, 15 per cent inhibition Mass.) (IO4counts/min/^ug) or of 100 fig. of orotic was observed; and 10 mg/kg of folinic acid com acid-6-C14 (Yolk Radiochemical Co., Chicago, 111.) pletely reversed the amethopterin-induced retar (1.7 X IO4 counts/min/Mg). One hour later the dation of purine synthesis. desired metabolites were isolated, and their spe An identical series of experiments was per cific radioactivities were determined as described formed measuring the effects of these agents on in the previous report (20). The folinic acid used the formation of DNA thymine. Orotic acid-6-C14 in these experiments was the calcium salt of the incorporation into DNA thymine was employed synthetic product and contained the inactive op as a measure of this metabolic route. The results tical isomer. Therefore, the concentrations spec obtained are presented in Table 3. Folinic acid had ified in the text are one-half the effective concen little effect on the rate of incorporation of labeled tration. orotic acid into DNA thymine but caused some RESULTS prevention of the inhibition produced by amethop Table 1 presents the effects of various doses of terin. The degree of reversal of amethopterin folinic acid on the amethopterin-induced prolonga inhibition was relatively insensitive to increasing TABLE1 EFFECTOFFOI.INICACJDONTHETUMOR-INHIBITOBYPROPERTIESOFAMETHOPTERIN A TreatmentControlFolinic dosage survival(days)10.5 «rt.(gm.)*+0.7+0.2 (mg/kg)2.5 +0.89.0 acidAmethopterinAmethopterin+folinic + 0.7 50.50.5+1.25 12.«±1.019.7 +2.4—1.1—0.8+2.2 +1.87.8 acidDaily + 1.3 0.5+2-5 8.9 + 1.2 0.5+5 7.5±0.6 +0.4+2.5 0.5+10Average 10.2 + 1.2Av. Drugs were administered intraperitoneally once daily for 6 consecutive days, beginning 24 hours after tumor implantation, with combination treatments given simultaneously. Controls are tumor-bearing animals given injections of isotonic sodium bicarbonate. Each figure represents the average ±standard error of results from ten to fifteen animals. * Average weight change from onset to termination of drug treatment. Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1962 American Association for Cancer Research. 104 Cancer Research Vol. 22, January 1962 concentrations of folinic acid. Furthermore, com bition of purine biosynthesis as did the simulta plete prevention of inhibition was not observed at neous administration of these two agents (Table doses of folinic acid up to 20 mg/kg. 5). Since folinic acid had been found to suppress the uptake of aminopterin by microbial cells (18, DISCUSSION 26), it was of interest to estimate the possible role Folinic acid itself is not the formyl donor in the of this phenomenon in the reversal of inhibition biosynthesis of purine and thymine nucleotides observed in the present study. The data presented but requires conversion to N'°-formyltetrahydro- in Table 4 indicate that folinic acid administered folic acid and to N6, N10-methenyltetrahydrofolic 30 minutes prior to or 30 minutes after the dose of acid to supply the cofactors required in the bio amethopterin completely reversed the growth in synthesis of purine nucleotides, and presumably a hibition by amethopterin. Furthermore, folinic conversion to N5, N10-methylenetetrahydrofolic to acid administered 30 minutes after a dose of 0.5 supply the one-carbon unit of thymidylic acid mg/kg of amethopterin brought about the same formation (3, 12, 19). The finding that folinic degree of reversal of amethopterin-induced inhi acid, at levels up to 20 mg/kg body weight, only TABLE2 EFFECTOFFOLINICACIDONTHEINCORPORATIONOFGLYCiNE-2-C"INTOADEMNEOFEHRLICHASCITESCELLS cent X cent X10-»NA 10-! AS TreatmentControlFolinic (mg/kg)5 of contro) of control adenine3.1+0.33.3±0.6 value106 adenine79.8 value105 3.984.1+8.0+ acidAmethopterinAmethopterin+folinic 100.50.5+2.5 4.4±0.30.7±0.42.0±0.31422365 100.56.918.8±6.050.9+ 1262464 acidDosage + 6.6 0.5+5 2.6 + 0.2 84 68.6 + 6.8 86 0.5+10Counts/min/Mmole3.6 + 0.7Per 116Counts/min/Mmole80.8 + 5.1Per 101 Tumor-bearing mice were each treated by intraperitoneal injection with the indicated dosage of amethopterin, folinic acid, or the simultaneous combination of amethopterin and folinic acid.