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The Synergistic Anti-Neoplastic Activity of Combinations of Mitomycins with Either 6-Thioguanine Or @.-F1uorouracip

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The Synergistic Anti-Neoplastic Activity of Combinations of Mitomycins with Either 6-Thioguanine Or @.-F1uorouracip CANCER RESEARCH VOLUME 25 OCTOBER 1965 NUMBER 9 The Synergistic Anti-neoplastic Activity of Combinations of Mitomycins with Either 6-Thioguanine or @.-F1uorouraciP ALAN C. SARTORELLI AND BARBARA A. BOOTH Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut SUMI@1ARY Mitomycin C or porfiromycin (methyl mitomycin) in combination with either 6- thioguanine or 5-fluorouracil produced synergistic inhibition of the growth of both Sar coma 180 and Lymphoma L1210 ascites cell neoplasms. i\Iitomycin C and porfiromy cm possessed the same therapeutic index in the L1210 lymphoma system, but mitomycin C was about 5 times more potent. Doses of mitomycin C that induced pro nounced inhibition of thymidine-3H incorporation into deoxyribonucleic acid (DNA) did not decrease the average DNA content of treated cells. The degree of inhibition of ribonucleic acid (RNA) synthesis by mitomycin C was less pronounced than that of DNA, and lysine-1-'4C fixation into residual protein was insensitive to the antibiotic. 6-Thioguanine induced a marked depression in the rate of incorporation of thymidine 3H and orotic acid-6-'4C into DNA and RNA, respectively. The combination of these agents produced subadditive to additive inhibition of the formation of nucleic acids and also caused inhibition of protein synthesis; incorporation of thymidine into DNA ap peared to be the metabolic path most sensitive to the combination of these agents. Measurement of thymidine kinase and thymidylate kinase activity in cells treated with these agents indicated that these enzymes were relatively resistant to the action of mi tomycin C ; however, some inhibition of thymidylate kinase activity was induced by the thioguanine treatment. It is hypothesized that these combinations inhibit cell reproduction by a complementary process involving DNA, the mitomycins acting to cross-link existing DNA molecules, thereby interfering with primer functions, and the anti-metabolites providing a more complete blockade of the synthesis of DNA by in hibition of specific enzymatic sites on the biosynthetic route. In previous reports from this laboratory (29, 30) it was cells (29). That the metabolic attack was indeed directed suggested that synergistic drug combinations might be against RNA was supported by the finding that the 2 drugs selected rationally by combining agents that bind and in in combination caused a subadditive depletion of the total activate cellular informational molecules (i.e., DNA and cellular RNA content ; presumably some fraction of RNA RNA) or enzymes with drugs capable of altering biosyn was decreased to a level which became limiting for cell thetic pathways leading to the formation of the damaged survival. polymers. This mechanism, termed “complementary in This rationale was extended further with DNA as the hibition,― was illustrated by the demonstration that corn “targetsite―(31). Mitomycin C or porfiromycin (methyl binations of ribonuclease and aetinomycin D produced mitomycin) was used to attack specifically the pre-existing synergistic inhibition of the growth of Sarcoma 180 ascites molecules of DNA, because these antibiotics have been 1 This investigation was supported by USPHS Research Grant indicated to function by cross-linking complementary CA-02817 from the National Cancer Institute and by Grants ACS strands of the DNA double helix and by inducing a de IN-31E-806 and ACS IN-31F-905 from the American Cancer Society. polymerization of the DNA itself (39). The mitomycins Received for publication February 10, 1965. were combined with either 6-thioguanine or 5-fluorouracil, 1393 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1965 American Association for Cancer Research. 1394 Cancer Research Vol. 25, October 1965 anti-met abolites that, following activation , possess the ving 50 days and the @)ercentage of regressions of tumor potential to retard DNA synthesis presumably at the nu growth were unequivocally greater than the sum of these cleotide level. This approach gains support from the effects produced by each drug alone. All mice surviving finding that cytosine arabinoside, an agent that also in over 50 days were calculated as 50-day survivors in deter hibits DNA formation (6), caused synergistic inhibition of mination of the average survival time. neopla.stic cell growth in combination with porfiromycin Drug-induced metabolic effecls.—Anirnals bearing 6-day (8). The data in this report describe the synergistic growths of Sarcoma 180 a.scites cells were each given a growth-inhibitory effects exhibited by combinations of the single i.p. dose of either mitornycin C, 6-thioguanine, or a mitornycins with 6-thioguanine or 5-fluorouracil on tratis combination of the 2 drugs. At selected time intervals planted rodent neopla.srns and provide evidence supporting thereafter, either 200 @gof thyniidine methyl-3H (5.4 X the hypothesis that these combinations may function to 10@c))ni/@Lg), 100 @gof orotic acid-6-'4C hydrate (2.1 X destroy these cells by a complementary process that in 10@cJ)fli/@ug), or 150 @hgof DL-lysine-1-'4C monohydrate volves the nucleic acids. (2.2 X 10@cpm/@g)were administered by i.p. injection to each mouse and were allowed 1 hr for metabolic utilization. MATERIALS AND METHODS In experiments involving the incorporation of thymidine Tumor lransplantalion.—Experi metits were performed 3H and orotic acid-6-'4C into nucleic acids, sodium nude on 9- to 11-week-old female Ha/ICR Swiss mice (A. R. ates were isolated by the method of Tyner ci al. (41) and Schmidt Company, Madison, Wisconsin) bearing Sarcoma hydrolyzed with 70 % perchloric acid for 1.5 hr (22). Ex @@ 180 a.scites cells, male C57BL x DBA F1 (hereafter tracts were desalted on charcoal columns, and the desired called BDF1) Ii1iC(@(Cumberland View Farms, Cumber pyrirnidine components of the nucleic acids were purified land, Tennessee) bearing L1210 lymphorna cells. Trans and analyzed as described by Danneberg et al. (7). After plantation of fl€@O))[email protected] out by withdrawing exposure of cells to lysine-1-'4C, residual protein was iso peritoneal fluid from a donor mouse bearing a 7-day tumor lated and analyzed as previously described (3). Radio growth. The suspension was centrifuged for 2 miii activity was measured with a Packard Tri-Carb liquid (1600 x g), the supernatant peritoneal fluid was decanted, scintillation spectrometer in all cases except those in which a 10-fold dilution with isotonic saline was made, and 0.1 the radioactivity of residual protein was measured; in the ml of the resulting cell suspension was injected i.p. into latter a Nuclear-Chicago gas flow counter equipped with a each aninial. Microniil window was employed. The PhosPhor solution Drug 1herapy.—F@orany one experiment, animals were used in the liquid scintillation counting contained 100 mg distributed into groups of 5—10mice of comparable weight of p-bis [2-(5-phenyloxazolyl)] benzene and 8 gm of 2,5- and maintained throughout the course of the experiment diphenyloxazole dissolved in 2 liters of toluene and 1 liter on Purina laboratory chow and water ad libiluin. Ex of absolute ethanol. periments with more thati 5 mice were performed at least The DNA and RNA content of the cells was measured 2 times, and the results obtained were averaged. by pentose analyses (33) in which deoxyadenosine and Drugs were administered by i.p. injection once daily for adenosine, respectively, were the standards. Cell volumes 6 consecutive days beginning 24 hr after tumor implanta were measured in hematocrit tubes by determining the tion, and combination treatments were administered simul volume occupied by a known number of cells. The cell taneously. 1\Iitomycin C, porfiromycin, and 5-fluorouracil number was determined with a Coulter particle counter, were administered iii solution in isotonic saline, whereas model A. 6-thioguanine was solubilized in isotonic saline with sodium Enzyme extracts were prepared from 6-day growths of hydroxide and adjusted to pH 7—8with hydrochloric acid. Sarcoma 180 ascites cells ; all procedures were carried out Solutions of mitornycin C and porfiromycin were freshly at 4°C. Cells were treated for 3 miii with 4 volumes of prepared every 3—fldays; during this time the antibiotics 0.2 % sodium chloride to lyse erythrocytes ; subsequently, were protected from light and were stored at 4°C. All an equal volume of 1.6 % sodium chloride was added and drugs were administered in volumes of 0.25—0.5ml. Con the cells were collected by centrifugation at 1000 X g. Af trols given injections of comparable volumes of saline were ter 1 wash with isotonic saline, the cells were suspended in included in each experiment. Mice were weighed during 4 volumes of 0.05 Mtris (hydroxymethyl) aminomethane the course of the experiment, and the percentage of change buffer, 1)11 7.5, containing 0.05 Mmole of thymidine/mi. in body weight from onset to termination of therapy was The cell suspension was disrupted with a Branson sonifier used as an indication of drug toxicity. at 8 amp. by one 30-sec burst; tubes containing the cell Evaluation with a.scitic iieoplasnis of combinations of suspensions were immersed in a methanol-ice bath during drugs as synergists could not be based solely on the pro sonication. The sonicates were centrifuged at 105,000 X longation of the survival time afforded by drug treatments, @1for 1 hr, and the supernatant fraction was collected. The since therapy which Produces a significant number of re protein content of the extracts was determined by the bi gressions of tumor growth (complete eradication of neo uret method (18) with twice recrystallized bovine albumin plastic cells) results in difficulty in expressing meaningful as the standard. Thymidine kina.se, thymidylate kinase, average survival times. Therefore, 2 additional criteria and thymidylate nucleotidase activities were measured by the number of animals surviving 50 days and the number incubating the following mixture at 37°C: either thymi of regressions of tumor growth—also were used to assess dine-3H, 0.1 @mole(2.9 X 10@cpm) or thymidylate-2-'4C, the effectiveness of drug treatments.
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