Effects of Folinic Acid on Amethopterin-Induced Inhibition of the Ehrlich Ascites Carcinoma ALAN C

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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 amethopterin'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

Survival experiments.â€”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.â€”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 Â«rt.(gm.)*+0.7+0.2 (mg/kg)2.5

+0.89.0

acidAmethopterinAmethopterin+folinic + 0.7 50.50.5+1.25 12.Â«Â±1.019.7 +2.4â€”1.1â€”0.8+2.2

+1.87.8

acidDaily + 1.3 0.5+2-5 8.9 + 1.2 0.5+5 7.5Â±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 Â±standard error of results from ten to fifteen animals. * Average weight change from onset to termination of drug treatment.

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'Â°-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-Â»NA 10-! AS TreatmentControlFolinic (mg/kg)5 of contro) of control adenine3.1+0.33.3Â±0.6 value106 adenine79.8 value105

+3.984.1+8.0 acidAmethopterinAmethopterin+folinic 100.50.5+2.5 4.4Â±0.30.7Â±0.42.0Â±0.31422365 100.5 +6.918.8Â±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. One hour later, 100.//g. of glycine-2-C14(IO4counts/min/^g} was injected per mouse and allowed to be incorporated for 1 hour. Each value represents the average + standard error of results from the separate analyses of ascites cells from four to eight animals.

TABLE 3 EFFECTOFFOLIMCACIDONTHEINCORPORATIONOFOROTICAciD-6-C14 INTODNATHYMINEOFEHRLICHASCITESCELLS

centof TreatmentControlFolinic (mg/kg)5100.50.5+2.50.5+50.5+100.5+20Counts/min/MmoleX10-2DNAthymine2.1+0.21.5Â±0.22.0controlvalue71951948436757

acidAmethopterinAmethopterin +0.20.4 +0.081.0 + folinic acidDosage +0.30.9 +0.21.4 +0.11.2Â±0.2Per

Tumor-bearing mice were each treated by intraperitoneal injection with the indi cated dosage of amethopterin, folinic acid, or the simultaneous combination of ameth opterin and folinic acid. One hour later, 100 ng. of orotic acid-6-C14 (1.7 X IO4 counts/min/jig) was injected per mouse and allowed to be incorporated for 1 hour. Each value represents the average Â±standard error of results from the separate analyses of ascites cells from four to fifteen animals.

partially reversed the inhibition of thymine forma opterin. Such a mechanism would result in an tion by amethopterin in Ehrlich ascites cells is in exceedingly sensitive reaction and may account agreement with previous findings in other systems. for the inability of folinic acid to completely re For example, folinic acid has been shown to pre verse the antifolic-induced inhibition of orotic vent, in part, the reduction of formate-C14 fixation acid incorporation into thymidylic acid. It is also into DNA of rabbit bone marrow by aminopterin conceivable, however, that the inability of folinic (23) and to partially overcome the inhibitory acid to completely prevent the amethopterin- action of aminopterin on the conversion of uracil- induced inhibition of DNA thymine formation 2-C14 deoxyribonucleoside to DNA thymine of may be the result of an enzymatic block other than chick embryo (6). Inhibition in the chick embryo folie reductase. Since the methylation reaction system was not completely overcome, even with a involved in the formation of thymidylic acid is ratio of folinic acid to aminopterin of 100:1. It is not directly inhibited by amethopterin (15), it is interesting to note that in the current experiments doses of folinic acid which completely prevented TABLE 5 the amethopterin-induced depression of purine EFFECTOFADMINISTRATIONSEQUENCEOFFOLINICACID ONTHEAMETHOPTERIN-INDUCEDINHIBITION biosynthesis only partially prevented the inhibi OFFURINEBIOSYNTHESISDEA'oro tion of orotic acid incorporation into DNA thy mine by amethopterin. Evidence is available which indicates that tetrahydrofolic acid partici- CoU.\T8/MIN/^MOLEX10"2 TREAT- TABLE 4 MENTControlAmethopterinAmethopterin .niellili,-8.6Â±0.20.6Â±0.042.adenine84.5Â±2.818.2 EFFECTOFADMINISTRATIONTIMEOFFOLINICACID ONTHETUMOR-INHIBITORYPROPERTIES OFAMETHOPTERIN +3.759.5 + simultn ne- Awt. acidAmethopterin+ous folinic 7 + 0.52.7Â±0.2AS +4.051.7Â±3.1 TreatmentControlAmethopterinAmethopterin+folinicsurvival (gn>.)*â€”1.0â€”1.8+0.4+1.5+ (dayÂ«)10.0Â±0.818.0Â±1.57.5 subsequent folinic acidNA

Tumor-bearing mice were each treated by in tra peritoneal injection with 0.5 mg/kgof amethopterin, 0.5 mg/kg of ameth acid opterin simultaneous with 5.0 mg/kg of folinic acid, or fAmethoptcrin+folinic(simultaneous) +1.89.0 0.5 mg/kg of amethopterin and 5.0 mg/kg of folinic acid 80 minutes later. Ninety minutes after the amethopterin, 100 acid Mg.of glycine-2-C14 (IO4 counts/min//ig) was injected per mouse (prior)ÃŽAmethopterin-)-folinic +1.09.1+1.0Av. and allowed to be incorporated for 1 hour. Each value repre sents the average + standard error of results from the separate acid analyses of ascites cells from four animals. (subsequent)!Average 1.6 possible that a sensitive site occurs on the pathway Amethopterin at a level of 0.5 mg/kg and folinic acid at by which folinic acid is converted to the coenzy- 5.0 mg/kg were administered intraperitoneally once daily for 6 consecutive days, beginning 24 hours after tumor implanta matic form functioning in the methylation of tion. Controls are tumor-bearing animals given injections of uridine nucleotides. The finding that formate in isotonic sodium bicarbonate. Each figure represents the average +standard error of results from ten animals. corporation into DNA thymine was more sensitive * Average weight change from onset to termination of to folie acid than was the incorporation into pu- drug treatment. rines (20, 23, 24) might also be the result of either t Amethopterin and folinic acid injected simultaneously. of these two alternatives. Ã•Folinic acid injected 30 minutes prior to amethopterin. The quantity of folinic acid required to reverse Â§Folinic acid injected 30 minutes after amethopterin. the growth inhibition by the folie acid antagonist was sufficient to prevent the amethopterin-induced pates in the biosynthesis of thymidylic acid by retardation of purine synthesis. This is in contrast acting both as a carrier for the one-carbon frag to results obtained with leukemia Ll210-bearing ment and as a reductant for the conversion of the mice when 3',5'-dichloroamethopterin was used as hydroxymethyl group to the methyl group, being an inhibitor (21). About 100:1 parts of folinic acid oxidized in the process to dihydrofolic acid (5, 13, were required to counteract the dichloroamethop- 15). To regenerate tetrahydrofolic acid would re terin inhibition of formate-C14 incorporation into quire the continued action of dihydrofolic acid acid-soluble adenine of leukemic spleens, whereas reducÃase,an enzyme markedly inhibited by ameth the therapeutic effect of daily doses of 40 mg/kg

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Alan C. Sartorelli, Herbert F. Upchurch and Barbara A. Booth

Cancer Res 1962;22:102-106.

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