Dietary Influence of Tyrosine and Phenylalanine on the Response of B16 Melanoma to Carbidopa-Levodopa Methyl Ester Chemotherapy1

[CANCER RESEARCH 42, 3056-3063, August 1982] 0008-5472/82/0042-OOOOS02.00 Dietary Influence of Tyrosine and Phenylalanine on the Response of B16 Melanoma to Carbidopa-Levodopa Methyl Ester Chemotherapy1

Gary G. Meadows,2 Herbert F. Pierson, Rokia M. Abdallah, and Pankaj R. Desai

College of Pharmacy. Washington State University. Pullman, Washington 99164

ABSTRACT tumor systems (7, 20, 41-44, 48, 49) and recently in the treatment of human melanoma patients (45, 46). One analog, The effect of dietary tyrosine and phenylalanine on survival levodopa methyl ester, both alone and in combination with the of adult female C57BL/6 x DBA/2 F, mice bearing i.p. slow- dopa decarboxylase inhibitor, benserazide, is effective in the growing, moderately pigmented and fast-growing, highly pig- pigmented B16 melanoma system (41). In vitro observations mented B16 melanoma tumors was studied alone and in com suggest that this antitumor activity results from the in situ bination with carbidopa-levodopa methyl ester chemotherapy. generation of reactive quinone intermediates that preferentially These studies tested three different diets: a natural product inhibit DMA synthesis (47). diet containing 1.09% phenylalanine and 0.64% tyrosine (com The in vivo growth of B16 melanoma is inhibited by depletion mercial diet); a chemically defined crystalline amino acid diet of tyrosine and/or phenylalanine. Restriction of these 2 amino containing 0.6% phenylalanine and 0.3% tyrosine (purified acids, either through dietary deficiency (5, 6, 8, 16, 18) or by diet); and a nutritionally deficient chemically defined diet con administration of specific degrading enzymes (10, 11, 21), taining 0.08% phenylalanine and 0.04% tyrosine (deficient inhibits growth of both experimental and human melanomas. diet). Mice received carbidopa (100 mg/kg) and levodopa Tyrosine depletion in vitro has a cytostatic effect on B16 methyl ester (1000 mg/kg) i.p. daily for 12 days. melanoma cells and is a strong inhibitor of RNA synthesis (9). The median survival of mice bearing the slow-growing tumor Dietary restriction of tyrosine and phenylalanine also alters averaged 8 days longer than that of mice bearing the fast- host immune responses (3, 17, 23). The exact mechanism growing tumor. Median survival increased by 42% (slow-grow whereby dietary restriction of tyrosine and phenylalanine cre ing tumor) and by 30% (fast-growing tumor) in mice maintained ates this inhibitory environment for tumor growth remains un on the deficient diet. Drug treatment significantly increased certain. survival in mice maintained on the purified and deficient diets Dietary tyrosine and phenylalanine and perhaps other large but was relatively ineffective in mice maintained on the com neutral amino acids may actually modulate the antitumor activ mercial diet regardless of the tumor growth characteristics. ity of levodopa. Consuming high-protein diets consisting of The largest increases in median survival of 61 % (slow-growing high levels of tyrosine and phenylalanine impairs both the tumor) and of 78% (fast-growing tumor) occurred in the treated adsorption and therapeutic effect of levodopa in human Parkin- mice maintained on the deficient diet. Plasma tyrosine and sonism patients (4, 15), whereas low-protein diets tend to phenylalanine levels decreased by 33 and 21%, respectively, potentiate and stabilize the therapeutic effects of this drug (4, in mice maintained on the deficient diet and were unaffected 15, 22). Although the mechanism responsible for the impaired by drug treatment. Tumors were 60% smaller in mice main response in patients consuming the high-protein diets is not tained on the deficient diet compared to mice maintained on definitively known, it is well established that the aromatic amino the purified diet. acids tyrosine, phenylalanine, levodopa, and tryptophan and Drug treatment resulted in decreased food intake and weight the branched-chain amino acids leucine, isoleucine, and valine loss in all tumor-bearing dietary groups. Restricting food intake compete for absorption at both the blood-brain barrier and at in untreated tumor-bearing mice to the amounts consumed by neuronal membranes. The apparent competition results be the drug treatment groups resulted in a parallel loss in body cause these amino acids share a common uptake mechanism weight but no significant alteration in median survival. These (13, 24, 50). The antitumor activity of levodopa is dependent data show that concomitant dietary tyrosine-phenylalanine re upon selective incorporation into melanoma cells (41, 42, 49), striction enhances the antitumor activity of carbidopa-levodopa and amino acid competition may interfere with the effectiveness methyl ester against B16 melanoma. of this drug. The importance of large neutral amino acids to the antitumor INTRODUCTION effect of various other drugs is also known. The uptake and cytotoxicity of L-phenylalanine mustard against murine L1210 Both levodopa treatment and tyrosine and/or phenylalanine leukemia cells are inhibited competitively by leucine (39). The restriction have inhibitory effects on the growth of malignant antitumor activity of azaserine is more effective in animals melanoma. Levodopa and related catechol compounds show maintained on an isoleucine-deficient diet (33). The amino acid significant antitumor activity in a variety of experimental animal analog, p-fluorophenylalanine, is totally dependent on concur rent dietary restriction of phenylalanine for antitumor activity ' These studies were supported in part by funds provided to Washington State against BW7756 hepatoma and C3HBA mammary adenocar- University through the NIH BiomÃ©dical Research Support Grant and by funds cinoma tumors (30). Phenylalanine imbalance as an adjunct to from the National Cancer Institute Grant CA 26533 and the American Cancer non-amino acid analog therapy greatly enhances the response Society Grant PDT-166. 2 To whom requests for reprints should be addressed. of human patients receiving combination chemotherapy with Received May 1, 1981 ; accepted May 3, 1982. mitomycin C, 5-fluorouracil, 1-/J-D-arabinofuranosylcytosine,

3056 CANCER RESEARCH VOL. 42

and toyomycin and in individuals receiving single-drug therapy dextrose, and 40.96% sucrose as sources of carbohydrate, and 5% with 6-mercaptopurine or steroids (19). cellulose as a source of fiber. Five % HMW Salts Modified to Meet In this study, we examine the combined antitumor activity of NRC Rat and Mouse Requirements was added as a source of minerals. levodopa methyl ester administered with the dopa decarbox- The salt mixed consisted of (g/kg): dibasic calcium phosphate, 456; calcium carbonate, 207.917; sodium chloride, 112; monobasic potas ylase inhibitor, carbidopa, combined with dietary restriction of sium phosphate, 154; magnesium carbonate, 25.2; magnesium sulfate, tyrosine and phenylalanine. This approach offers the advan 16; ferric phosphate, 20.8; potassium iodide, 0.08; manganese sulfate, tage of utilizing different antitumor mechanisms concomitantly 4.7; sodium fluoride, 0.12; aluminum potassium sulfate, 0.16; cupric and avoids the potential competition for uptake into melanoma sulfate, 0.88; zinc carbonate, 2.14; and sodium selenite, 0.003. A cells between dietary amino acids and the drugs. Since carbi 2.2% Diet Fortification Bio-Mix No. 20315 was added as a source of dopa is a competitive inhibitor of tyrosine aminotransferase vitamins and consisted of (g/kg): vitamin A, 4.5 (200,000 lU/g); (37) and since administration of this drug can result in a 2- to vitamin D, 0.25 (400,000 lU/g); a-tocopherol, 5.00; ascorbic acid, 3-fold increase in endogenous serum and tissue levels of 45.00; /-inositol, 5; choline chloride, 75; menadione, 2.25; p-amino- tyrosine (1, 51 ), limitation of dietary tyrosine and phenylalanine benzoic acid, 5; niacin, 4.5; riboflavin, 1.00; pyridoxine-HCI, 1.00; thiamine-HCI, 1.00; calcium pathothenate, 3.00; biotin, 0.020; folie should enhance the effect of the antitumor drug. acid, 0.009; and vitamin Bi2, 0.00135. The diet also contained 0.015% a-tocopherol acetate and 2% sodium bicarbonate for preservation of MATERIALS AND METHODS labile constituents and pH control. The deficient diet was prepared by reducing the tyrosine and phe Chemicals. All chemicals and drugs used in this study were reagent nylalanine content of the purified diet to 0.08% phenylalanine and grade. Levodopa methyl ester was purchased from Sigma Chemical 0.04% tyrosine. Glycine and glutamic acid were altered to make this Company, St. Louis, Mo. Carbidopa was a gift from Merck Sharp and diet isonitrogenous to the purified diet. Both diets were stored in the Dohme, West Point, Pa. dark in tightly sealed containers at 4Â°to minimize deterioration (35) Mice. Specific-pathogen-free female C57BL/6 x DBA/2 F, (here and used within 90 days after purchase. All diets were administered in after called B6D2F,) mice were purchased from Harlan/Sprague-Daw- pelleted form, and fresh pellets were offered daily. ley, Madison, Wis., at 4 to 6 weeks of age. Adult female mice at 14 to Amino Acid Analyses. Food was removed from mice at least 8 hr 16 weeks of age and averaging 21.1 Â±1.2 (S.D.) g were used for all prior to collection of blood. Blood was collected at the same time (9 experiments. The mice were maintained ad libitum on a locally manu a.m.) of day to avoid changes in plasma amino acid levels due to factured natural product diet containing 18.7% crude protein, 2.6% diurnal variation (12). Mice were bled by the orbital technique (25). crude fat, and 6.7% crude fiber until distributed into experimental Blood from groups of 4 to 6 mice was collected in heparinized capillary dietary groups. Mice were accustomed to the test diets for at least 2 tubes, transferred to chilled microcentrifuge tubes, and centrifuged. A weeks before antimelanoma experiments. known weight of plasma was processed for amino acids using the The animal facility is accredited by the American Association for perchloric acid method of Saifer (31). The processed samples were Accreditation of Laboratory Animal Care and conforms to their stand-' lyophilized at â€”50Â°ina VirTis lyophilizer and stored at -70Â° prior to ards for the care and use of laboratory animals. The mice were housed in separate quarters where temperature was controlled to 24 Â±1Â°and analysis on a Beckman Model 121 MB amino acid analyzer. Norleucine was added as an internal standard. humidity was controlled to 53 Â±5%. There were 6 to 10 air changes/ Tumor. B16 melanoma tumors from 2 different sources were used hr, and the mice were kept on a 12-hr-light, 12-hr-dark cycle with light in these studies. Results with a moderately pigmented, slow-growing from 6 a.m. to 6 p.m. Experimental mice were pair-housed in sus B16 melanoma tumor obtained through the courtesy of Dr. Vernon pended stainless steel cages (20 x 25 x 18 cm) with mesh floors and Riley, Fred Hutchinson Cancer Research Center, Seattle, Wash., are sides unless otherwise indicated. Each cage was also provided with presented in Tables 1 and 2 and in Chart 1. Results with a faster- Nestlet bedding (Aneare Corporation, Manhasset, N. Y.). Food intake growing, highly pigmented tumor obtained from the Mason Research was determined daily to an accuracy of Â±0.5 g, which allowed for Institute, Worcester, Mass., are presented in Table 3 and Chart 2. This slight inefficiencies in food retrieval. Small food particles were easily tumor was used for determining the effect of food restriction on survival retrieved from the Nestlet bedding and from paper placed under each of tumor-bearing mice. Both tumors were maintained in vivo as s.c. cage. Water intake was measured in glass cylindrical drinking tubes transplants. All injected tumor suspensions, prepared according to the calibrated to Â±1ml (BioServ, Inc., Frenchtown, N. J.). method of Fidler ef al. (14), were greater than 98% viable by trypan Test Diets. LabBlox Sterilizable Animal Diet commercial diet was blue exclusion tests. Mice received 106 viable cells i.p. in a fixed obtained from Allied Mills, Chicago, III. This standard natural-product volume of 0.1 ml. Assay of tumor-bearing mice for lactate dehydrogen- laboratory chow consists of 24% crude protein, 4% crude fat, and ase (2) suggested the presence of the ubiquitous lactate dehydrogen- 4.5% crude fiber and provides 3.86 kcal/g of food. This diet contains ase-elevating virus, but its actual presence was not confirmed. Al 1.09% L-phenylalanine and 0.64% tyrosine, which is of particular though this virus is known to be present in many transplantable tumors significance to this study. (29), it does not significantly affect the growth rate of the pigmented The synthetic diet (purified diet) was composed of crystalline amino B16 melanoma (28) but can cause immunological and other alterations acids and was prepared by BioServ, Inc. It is adequate for growth, maintenance, and reproduction of B6D2Ft mice.3 The diet is equivalent in the host (26, 27). Antimelanoma Experiments. Mice receiving the commercial diet in protein to an 11.8% casein diet, provides approximately 4.0 kcal/g, were stabilized on the diet for 3 weeks before tumor inoculation. All and is similar in amino acid composition to the diets described by other mice were initially stabilized for 1 week on the purified diet. One- Bounous and Kongshavn (3) and by Theuer (36). The diet contained half of the animals were continued on this diet, and one-half were 0.6% L-phenylalanine, 0.3% L-tyrosine, 0.5% L-isoleucine, 0.8% L- placed on the deficient diet for an additional 2-week period. Mice were leucine, 0.9% L-lysine, 0.4% L-methionine, 0.5% L-threonine, 0.7% L- allowed free access to all diets throughout the experiments except for valine, 0.15% L-tryptophan, 0.3% L-histidine, 0.21% L-arginine, 0.2% those mice involved in the diet restriction study. Water was freely L-cystine, 0.53% L-alanine, 1.23% L-aspartic acid, 0.23% L-glycine, available to mice at all times. Test mice were given injections of B16 1.96% L-proline, 1.04% L-serine, and 4.8% L-glutamic acid. The diet melanoma after the stabilization period on Day 0. Treated groups also contained 10% corn oil as a source of fat, 6.0% cornstarch, 15% received daily carbidopa-levodopa methyl ester therapy beginning 24 hr following tumor inoculation and continuing for 12 days. Carbidopa ' Unpublished observations. (100 mg/kg) was administered 90 min prior to injection of levodopa

AUGUST 1982 3057

Table 1 Effect of diet and drug treatment on survival and tumor weight in mice bearing the slow-growth B16 melanoma tumor survival sur in tumorwt(g)c7.5 DietCommercialPurifiedDeficientGroup8Untreated(days)37 vival(days)364131394450Increasesurvival6(%)1632264261Mean (22)"Treated Â±1e40 Â±0.56.7 (12)Untreated Â±132 Â±0.98.5

(26)Treated Â±140 Â±0.77.9 Â±2f46 (16)Untreated Â±0.83.3

(22) Â±2 Â±0.59 Treated (16)Mean 53 Â± 2'Median 3.4 Â±0.59 a Untreated groups are composed of 0.9% NaCI solution-injected and uninjected mice. The groups are combined since statistical analysis indicated no significant difference in the mean survival. Treated groups were given injections of drug from Days 1 to 12 post-tumor inoculation as outlined in "Materials and Methods." /Median survival of dietary untreated or treated group \ % of increase = - 1 x 100 \ Median survival of purified diet-untreated group / c Tumor weight determined at necropsy in animals that died from tumor, mean Â±S.E. d Numbers in parentheses, number of mice in each group. e Mean Â±S.E. ' Values significantly different from untreated mice within the same dietary group (p < 0.01). 9 Significantly different from all commercial and purified dietary groups (p < 0.01).

Table 2 Food consumption in drug treated non-tumor-bearing and tumor-bearing mice inoculated with the slow- growing tumor Food consumption was determined daily and averaged over the specified periods. Initial consumption was determined over a 7-day period before tumor inoculation and/or drug injection. Values for consumption during the 12-day treatment period and during the 10-day posttreatment period in tumor-bearing and non- tumor-bearing mice receiving daily i.p. injections of drug are presented. Each purified and deficient dietary tumor-bearing group contained 16 mice, and the commercial dietary group contained 12 mice. All non- tumor-bearing groups contained 10 mice. Mean food consumption (g)

Treatment period Posttreatment period

DietarygroupCommercial Â±0.1b Â±0.1 Â±0.1 Â±0.1 Â±0.1 Purified0 2.7 Â±0.1 1.6 Â±0.1 2.6 Â±0.1 2.5 Â±0.1 2.3 Â±0.1 2.1 Â±0.1aNontumor3.2 Deficient0Initial3.4 2.7 Â±0.1Tumor32.52.0 Â±0.1Nontumor3.32.7 Â±0.1Tumor3.7 2.7 Â±0.1 Significantly different from initial food consumption within dietary group (p < 0.001). 6 Mean Â±S.E. 0 Significantly different from commercial diet group (p < 0.001). methyl ester (1000 mg/kg). Both agents were prepared daily in 0.9% more than 2 means were made using the least-significant-difference NaCI solution and injected i.p. in a volume equal to 1% of mouse body test after analysis of variance (34). weight. Untreated groups were given injections of 0.9% NaCI solution. SomeIn order dietary to assure and qualitytreated control, groups someconsisted mice of were both not tumor-bearing given injections. and RESULTS """ non-tumor-bearing mice. The antitumor response was determined from Slow-growing Moderately Pigmented Tumor. The data pre- the mean and median survival time. sented Â¡nTab|e 1 show the effect Qf d|et ar)d dmg treatment on Diet Restriction Study. All mice were single housed in suspended ^.^ Qf mÂ¡ceÂ¡ |anted wj,h the s|ow.growing tumor. Three stainless steel cages and maintained on the purified diet until their .. .. weight stabilized. One-half were continued on this diet, and one-half 9rouPs were maintained on commercial laboratory chow, the were placed on the deficient diet for an additional 2 weeks. Untreated, Purified diet- and the deficient diet. The similarity between the treated, and restricted subgroups each containing 10 mice were formed mean and median survival times indicates that the tumor grew within the dietary groups and inoculated with the fast-growing tumor uniformly in both untreated and treated dietary groups. Diet on Day 0. The untreated and treated subgroups were allowed free alone altered survival. Untreated mice maintained on the corn- access to the diets and were handled as outlined under "Antimelanoma mereiai diet showed a slight (16%) increase Â¡nmedian survival Experiments." Beginning on Day 0, food intake was limited to 1.6 Â± compared to those maintained on the purified diet. A 42% 0.1 (S.E.) g for 12 days and to 2.5 Â±0.1 g thereafter Â¡nthe restricted increase occurred in control animals fed the deficient diet subgroup receiving the purified diet; intake was limited to 2.0 Â±0.1 g a|one Drug treatment increased median survival in all dietary throughout the experiment in the restricted subgroup receiving the wjth the def|cÂ¡ent dj exhibiting the greatest deficient diet. These levels were selected to parallel the intake of the ,-â€žâ€ž,.^ *â€¢^ , , , *!L. , -Â¿u- drug-treated mice bearing the slow-growing tumor (Table 2). Both lncrease <61 %>' Statistical analysis of the mean survival within restricted subgroups were given injections of 0.9% NaCI solution from dietarV 9rouPs indicated no therapeutic effect in mice fed the Days 1 to 12 commercial diet but significant therapeutic effects in both the Statistical Analysis. Significance between 2 means was determined purified and deficient dietary groups, by using Student's f test. All multiple pairwise comparisons between The weight changes observed Â¡ntreated and untreated tu-

3058 CANCER RESEARCH VOL. 42

29 The most profound changes in weight resulted from the drug 28 treatment, and both tumor-bearing and non-tumor-bearing 27 mice lost weight in all dietary groups. Tumor-bearing mice 26 25 maintained on the purified diet lost the most weight. Mice fed 24 the purified and commercial diets regained the weight after 23 drug treatment was stopped. Non-tumor-bearing mice retained 22 21 weight at a faster rate than did tumor-bearing mice. Mice fed 20 the deficient diet were unable to regain fully the weight lost 19 during treatment regardless of tumor presence. LU 32 Mice maintained on the commercial diet consumed signifi CO 31 +1 30 cantly more diet than did mice maintained on the purified or CD 29 deficient diets before tumor inoculation (Table 2). The purified 28 and deficient groups, however, readily accepted the diets. The 27 26 tumor did not affect food consumption in non-drug-treated g 25 LU mice until the last 1 to 3 days before death when some mice 24 23 became anorectic. Drug treatment significantly reduced food 22 consumption in tumor-bearing mice by 41% in the purified O 21 dietary group and by 26% in the commercial and deficient o 20 m 19 dietary groups. Normal food consumption resumed in mice fed 18 the commercial and purified diets after discontinuing drug 23 therapy. In contrast, food intake remained depressed in tumor- 22 bearing mice fed the deficient diet. Drug treatment did not alter 21 20 food intake in any of the non-tumor-bearing groups. These data show that appetite suppression in the tumor-bearing groups is not related to drug toxicity. Water intake was similar in commercial and purified dietary groups regardless of tumor, tumor type, or drug treatment and averaged 4.3 Â±0.1 (S.E.) ml. Water intake increased in mice fed the deficient diet during the 14-day stabilization period but decreased when their weight stabilized. Prestabilization intake DAYS was 11.6 Â±1.5 ml and decreased to 4.2 Â±0.1 ml at stabili Chart 1. Effect of diet and drug treatment on body weight in non-tumor- bearing and tumor-bearing mice inoculated with the moderately pigmented slow- zation. Intake was unaffected by tumor, tumor type, or drug growing tumor. Mice were divided into 3 dietary groups. Group A was maintained treatment. on the commercial diet. Group B on the purified diet, and Group C on the Fast-growing Highly Pigmented Tumor Experiments and restricted diet. All mice were started on the diets 14 days before tumor inoculation at Day 0. Weight data before Day 0 are presented as the pooled mean of all Diet Restriction Study. The effects of drug treatment and of animals maintained on the diets before assignment to subgroups (O). Non-tumor- food restriction on the median survival of mice implanted with bearing mice consisted of a drug treatment subgroup (A) and a subgroup the faster-growing, highly pigmented tumor are presented in containing 0.9% NaCI solution-injected plus uninjected mice O. Tumor-bearing mice were similarly divided into drug treatment (A) and the 0.9% NaCI solution- Table 3 and Chart 2. Table 2 shows that the median survival injected plus uninjected (â€¢)subgroups; bars, S.E. Non-tumor-bearing mice for mice implanted with this tumor is 8 days less than that for contained 10 mice in the drug-injected subgroup and 20 mice in the 0.9% NaCI mice implanted with the slow-growing tumor (Table 1). Mice solution-injected plus uninjected subgroup. The number of tumor-bearing mice treated with drug were: Group A, 12; Group B, 16; and Group C, 16. The number maintained on the deficient diet again showed a significant receiving 0.9% NaCI solution or no injections were: Group A, 22; Group B, 26; increase in survival. Drug treatment increased survival in both and Group C, 22. The 0.9% NaCI solution-injected subgroup is combined with the uninjected group since they are not statistically different. Each non-tumor- the purified and deficient dietary groups. In general, the drug bearing and tumor-bearing uninjected subgroup contained 10 mice. Drug treat appears to be more effective against the faster-growing, more ment and 0.9% NaCI injections were given from Days 1 to 12. Arrows, first and highly pigmented tumor in both purified and deficient dietary last day of treatment. groups, and activity is enhanced by feeding the deficient diet. Drug treatment again was ineffective in mice maintained on the mor-bearing and non-tumor-bearing mice maintained on the 3 commercial diet (data not shown). Minimal or no increase in diets are presented in Chart 1. Both the purified and commer median survival resulted from food restriction. cial diets supported normal weight gain in non-tumor-bearing Even though the food intake of the diet-restricted and the mice. Mice fed the deficient diet initially lost weight, but their treatment groups were not different, the restricted groups lost weight stabilized before tumor inoculation. more weight (Chart 2). The weight loss in these groups gener The tumor consistently inhibited weight gain in mice fed the ally paralleled tumor-bearing groups given injections of the commercial and purified diets during the first 12 days after slow-growing tumor and treated with drug, except in mice on inoculation. We attribute the later increases in weight in these the purified diet. This group retained about 0.5 g more body dietary groups to tumor growth. Tumor-bearing mice fed the weight than did their treated counterparts inoculated with the deficient diet showed a slight decline in body weight during the slow-growing tumor. first 12 days after tumor inoculation. No increase in weight Protein (amino acid) and phenylalanine consumption before, occurred in this dietary group from tumor growth. Injection of during, and after the treatment interval for both drug-treated 0.9% NaCI solution did not affect weight gain in either tumor- and food-restricted mice in all studies is compared in Tables 4 bearing or non-tumor-bearing groups fed any of the diets. and 5. Table 4 illustrates that the mice maintained on the

AUGUST 1982 3059

Table 3 groups during the treatment interval. Mice maintained on the Effect of diet, drug treatment, and restricted feeding on survival and tumor weight in mice bearing the fast-growing B16 melanoma tumor deficient diet actually consumed more amino acids on a mg/ kg basis than did mice maintained on the purified diet during the treatment interval. The diet restriction study shows that the sur tumorwt decrease in amino acid intake in the purified and deficient (g)c9.0 DietPurifiedDeficientGroup3UntreatedRestrictedTreatedUntreatedRestrictedTreatedMeanvival(days)24 dietary groups does not increase median survival (Table 3). Â±1d22 Â±0.16.4 Â±1e32 Â±0.4'5.7 Additional studies in our laboratory indicate that increasing Â±2'34 Â±0.6f3.3 the protein equivalent of the purified diet from 11.8 to 23.2% by doubling the amounts of all amino acids except for phenyl- Â±229 Â±0.593.4 Â±3e41 Â±0.693.6 alanine and tyrosine does not alter survival or the antitumor Â±3'Mediansurvival(days)232431303041Increaseinsurvival6(%)435303078MeanÂ±0.49 response to drug.4 Even though amino acid intake was doubled, a Untreated and restricted groups received 0.9% NaCI solution and treated mice still lost a similar amount of weight when treated with the groups received drug from Days 1 to 12 after tumor inoculation on Day 0 as described in "Materials and Methods." Actual food consumption was 1.7 Â±0.1 drug. g from Days 1 to 12 and 2.6 Â±0.1 g thereafter in the purified diet-restricted Table 5 illustrates that an average phenylalanine consump group. Consumption was 1.9 Â± 0.1 g in the deficient diet-restricted group tion ranging from 77 to 130 mg/kg increases median survival throughout the experiment beginning on Day 1. Parallel consumption was ob served in the drug-treated groups. Untreated and treated animals were given free (deficient diet). Drug treatment decreases phenylalanine (and access to the diets. Each group contained 10 mice. tyrosine) consumption further, but these changes are not sta % of increase tistically different (p > 0.05). Other experiments indicate that Median survival of group the phenylalanine intake must be lower than 364 Â±10 mg/kg \Median survival of purified diet-untreated groi ' oup / before an increase in median survival occurs.4 This value is c Tumor weight determined at necropsy in animals that died from tumor, mean significantly lower than is the 488 Â± 17mg/kg consumption Â±SE. " Mean Â±S.E. observed in the slow-growing tumor-bearing group maintained " Not significantly different from untreated group. on the purified diet (p < 0.05). ' Significantly different from untreated mice within dietary group (p < 0.001 ). 9 Significantly different from all groups maintained on purified diet (p < 0.01) The effect of the 3 diets on plasma tyrosine and phenylala but not significantly different from each other. nine levels is presented in Table 6. Plasma tyrosine levels in non-tumor-bearing mice fed the commercial and purified diets did not differ significantly. Phenylalanine levels were consis 32 31 tently higher in mice maintained on the commercial diet. Con 30 sumption of the deficient diet for 7 days resulted in a 33% ^ 2Â» LU 28 decrease in plasma tyrosine and a 21 % decrease in phenylal CO 27 anine in non-tumor-bearing mice compared to the purified â€¢*â€¢'28 dietary group. These levels remain lowered for at least 70 days. 3 25 The presence of tumor significantly altered tyrosine and 5E" phenylalanine metabolism in mice fed the purified diet. Tyrosine Ã• *2* levels increased by 9% 7 days after tumor transplant but then LU 20 decreased by about 23% in mice bearing 14-day tumors. ^ 18 Plasma tyrosine was also reduced in mice fed the commercial Â»^ 18 and deficient diets, which bore 14-day tumors. Tumor-bearing animals maintained on the purified and deficient diets showed CUS reduced plasma phenylalanine levels at 7 days with no further alteration in the levels at 14 days. Phenylalanine levels were also lower in 14-day tumor-bearing mice fed the commercial LU IB 2 " diet. Drug treatment did not alter plasma tyrosine and phenyl iÂ« alanine. 16 14 Tumor weight differed among the dietary groups. Tumors 13 were weighed at necropsy after the mice died naturally from

-15 -7-30 8 12 22 the tumor. These data are presented as part of Tables 1 and 3. DAYS Average tumor weights were not different in mice maintained Chart 2. Body weight changes in diet-restricted and drug-treated mice bear on the commercial and purified diets as shown in Table 1. ing the fast-growing, highly pigmented B16 melanoma tumor. Mice were divided Tumor weights did not differ significantly between the drug- into 2 dietary groups and 3 subgroups. Group A received the purified diet, and Group B received the deficient diet. All mice were stabilized for 14 days on the treated and untreated groups receiving the slow-growing tu diet before tumor inoculation at Day 0. Weight data in non-tumor-bearing mice mor, but tumor weights were smaller in the treated group are presented as the pooled mean of all mice before assignment to individual receiving the fast-growing tumor and maintained on the purified subgroups (O). Tumor-bearing subgroups were ad libitum fed and 0.9% NaCI solution injected (â€¢);ad libitum fed and drug injected (A); and restricted fed and diet. Food restriction inhibited tumor growth in mice maintained 0.9% NaCI solution injected (â€¢);bars, S.E. The drugs and 0.9% NaCI solution on the purified diet and receiving the fast-growing tumor. were administered i.p. from Days 1 to 12 as outlined in "Materials and Methods." All subgroups contained 10 mice. Arrows, first and last days of treatment. Tumors were significantly smaller (p < 0.05) in mice main tained on the deficient diet than in any of the other dietary commercial diet clearly were not malnourished for protein. groups, but the tumors weighed the same in untreated, re Non-tumor-bearing mice maintained on the purified and defi stricted, and treated groups regardless of tumor type. cient diets consumed similar amounts of amino acids. In all diets, consumption decreased in the treated and restricted * Manuscript in preparation.

3060 AUGUST 1982

Table 4 Protein (amino acid) consumption in tumor-bearing mice during drug treatment and diet restriction Protein (amino acid) consumption was determined daily for individual mice and averaged over the specified intervals. Initial consumption was determined over a 7-day period before tumor inoculation. Values for the treatment interval reflect the mean consumption during a 12-day drug treatment period or during food restriction. The posttreatment interval reflects the mean consumption during the 12 days after the treatment interval. (mg/g)aDietCommercial0PurifiedDeficientGroup Protein (amino acid) consumption

interval*27.7 type)Treated(Tumor interval39.9 (slow)Treated Â±0.5d16.7 Â±0.969.6 Â±1.0"17.1

(slow)Treated Â±0.218.5 Â±0.310.5 Â±0.417.8 (fast)Restricted Â±0.1e16.3 Â±0.210.9 Â±0.216.2 (fast)Treated Â±0.318.4 Â±0.315.5 Â±0.619.2

(slow) Â±0.4 Â±0.6" Â±0.7' Treated (fast) 15.4 Â±0.1e 11.4 Â±O.l' 17.0 Â±0.2' 'FoodRestricted (fast)Initial35.717.7 Â±0.4Treatment13.5 Â±oV' gPosttreatment14.2 Â±0.86 consumption (g) during interval Mouse weight (g) during interval '' Values significantly different compared to initial consumption within the diet and group (p < 0.05). ' All values for commercial dietary group significantly different from all values for purified and deficient dietary groups (p < 0.05). " Mean Â±S.E. 8 Significantly different from treated (slow) and restricted (fast) groups within diet (p < 0.05). ' All values significantly different from each other within the treatment interval (p < 0.05). 9 Significantly different within the same group (tumor type) maintained on purified diet during the treatment period (p < 0.05).

Table 5 Phenylalanine consumption in tumor-bearing mice during drug treatment and diet restriction Phenylalanine consumption was determined daily for individual mice and averaged over the specified interval. Initial consumption was determined over a 7-day period before tumor inoculation. Values for the treatment interval reflect the mean consumption during the 12-day treatment period or during food restriction. The posttreatment interval reflects the mean consumption during the 12 days after the treatment interval. Values for tyrosine consumption (not shown) are equal to one-half those reported for Phenylalanine. (mg/kg)aDietCommercial0PurifiedDeficient9Group Phenylalanine consumption

type)Treated(tumor interval1259 interval1814 Â°847Â±206 Â±436488 Â±456867 (slow)Treated

(slow) Â±12 Â±17*' Â±21 Treated (fast)' 535 Â±10e1 940 Â± 7 950 Â±12 Restricted(fast)Treated 827 Â±15125 553 Â±13105 825 Â±31130

(slow) Â± 2 Â± 4 Â± 5 Treated (fast) 104 Â±1120 77 Â± 1 115 Â±196 5FoodRestricted (fast)Initial1621 Â± 3Treatment 91 Â± 2Posttreatment Â± consumption (g) during interval Weight (kg) during interval " All values for commercial dietary group significantly different from all values for purified and deficient groups. Values within dietary groups significantly different from each other (p < 0.05). c Mean Â±S.E. Values significantly different from initial and posttreatment interval (p < 0.05). e Significantly different from treated (fast) and restricted (fast) group during treatment interval (p < 0.05). ' Values during all intervals significantly different from treated (slow) and restricted (fast) groups (p < 0.05). 9 All values significantly different from all other dietary groups during all intervals (p < 0.05).

Mice showed no visible lung metastasis when observed at with the deficient diet-drug combination. The data establish necropsy in any of the groups. The mice may have been killed dietary phenylalanine and tyrosine as important variables in by the primary tumor before evidence of pulmonary metastasis determining the chemotherapeutic responsiveness to this drug could be detected. and further support a role for tyrosine and phenylalanine in melanoma growth. DISCUSSION Consideration of diet would be particularly important if this drug or perhaps other analogs become clinically useful in the In this study, we examined the concomitant use of dietary treatment of human melanoma patients. One way of insuring limitation of tyrosine and phenylalanine and of carbidopa-lev- control over tyrosine and phenylalanine levels would be odopa methyl ester chemotherapy in the treatment of B16 through providing the patient with parenteral nutritional support melanoma. The results indicate that host survival is enhanced restricted in tyrosine and phenylalanine content. Conventional

CANCER RESEARCH VOL. 42 3061

Table 6 Effect of diet, tumor, and drug treatment on plasma tyrosine and phenylalanine Plasma levels (nmol/ml)a

Time Tyrosine Phenylalanine on diet tumor (days)21282121287142128wiin(days)14714714Non-tumor-bearingmice68 ingmice59 bearingmice69 ingmice60 Â±3b(6)c66 CommercialdietPurified Â± 1(6)61 Â± 0(2)72 Â± 0(2)53

(9)48Â± 1e* dietDeficient Â± 2(9)44 Â± 2s(6)51 Â± 1(6)49 Â± 3'(2)46 Â± 0(2)37

diet9rme Â± 4(6)45 Â± 2(6)51 Â± 3 (6)Tumor-bear Â± 3 (6)Tumor-bear Â± 1 "(5)43 Â± 1(5)31 (1)Non-tumor- (1) a Tyrosine and phenylalanine levels were determined on pooled plasma samples obtained from groups of 4 to 6 mice for the total number of pooled samples analyzed. b Mean Â±S.E. c Numbers in parentheses, number of samples analyzed. d Purified diet non-tumor-bearing untreated versus commercial diet non-tumor-bearing untreated (p < 0.001). e Purified diet tumor-bearing untreated versus non-tumor-bearing untreated (p < 0.001). ' Significantly different from purified diet tumor-bearing untreated group with 7-day tumor. 9 All values significantly different from commercial and purified diet tumor-bearing and non-tumor- bearing mice (p < 0.001). h Deficient diet tumor-bearing versus non-tumor-bearing (p < 0.001).

parenteral nutritional support could interfere with the therapeu required for asparagine-sensitive tumors, only partial reduc tic response to levodopa therapy since the currently available tions in the plasma level of tyrosine and phenylalanine are protein hydrolysates and crystalline amino acid preparations necessary to achieve an antitumor response in the B16 mela contain high amounts of phenylalanine (40). In fact, blood noma tumor. Only minimal weight loss occurred in mice fed the levels of phenylalanine and other amino acids are elevated deficient diet, and their weight was stable before tumor inoc during infusion of these amino acid-containing solutions (40). ulation. The antitumor effect derived from this diet is clearly In our study, we observed a poor chemotherapeutic response not related to weight loss but probably is related to the plasma in mice maintained on the commercial diet, which contains level of tyrosine and phenylalanine available to the tumor. 1.09% phenylalanine and 0.64% tyrosine. The antitumor re These data further support an altered nutritional requirement sponse to drug also is inhibited in mice that are maintained on for tyrosine and phenylalanine in melanoma tumors. a diet similar to the purified diet but containing 2% phenylala Additional studies are in progress to determine the specific nine and 1% tyrosine.3 Although these data support dietary levels of tyrosine and phenylalanine required for the growth of phenylalanine and tyrosine as important modifiers of the drug B16 melanoma in vivo, to determine the extent of inhibition response, interference by other constituents cannot be ruled imposed by dietary phenylalanine and tyrosine on levodopa out. The fact that survival of tumor-bearing mice maintained on uptake into melanoma cells, and to establish the mechanism the commercial diet was longer than was survival of mice responsible for inhibition of tumor growth by tyrosine and maintained on the purified diet suggests that the commercial phenylalanine restriction. diet contains some tumor-inhibitory constituent, which may also interfere with drug activity. ACKNOWLEDGMENTS Weight loss as a side effect of drug treatment occurred in all dietary groups. Patients with Parkinson's disease treated with The authors are grateful to Judith Salmon and Kai Johnson for their expert technical assistance and to Dr. Craig Coon for his helpful comments regarding these drugs also show weight loss, which may be due to formulation of the chemically defined diets. appetite suppression (38) or to effects on metabolic rate (32). We observed no depression of appetite in non-tumor-bearing, REFERENCES drug-treated mice, but appetite was depressed in tumor-bear ing animals. Both groups still lost weight during drug treatment. 1. Airoldi. L., Watkins, C. J., Wiggins, J. F., and Wurtman, R. J. Effect of pyridoxine on the depletion of tissue pyridoxal phosphate by carbidopa. These data suggest that the anorexia in tumor-bearing animals Metab. Clin. Exp., 27: 771-779, 1978. is related to the tumor and not to the drug treatment. From the 2. Bergmeyer, H. U., Bernt. E.. and Hess. B. Lactic dehydrogenase. In: H. U. diet restriction study, it is clear that decreased food consump Bergmeyer (ed.). Methods of Enzymatic Analysis, pp. 736-740. New York: Academic Press, Inc.. 1965. tion and the accompanying weight loss does not significantly 3. Bounous. G., and Kongshavn, P. A. L. The effect of dietary amino acids on alter survival of tumor-bearing mice. Feeding mice the 23.2% immune reactivity. Immunology, 35: 257-266, 1978. protein equivalent diet, which eliminates protein-calorie mal 4. Cotzias, G. C., Papavasiliou, P. S., and Gellene, R. Parkinsonism and dopa. Trans. Assoc. Am. Phys., 81: 171-183, 1968. nutrition as a contributing factor to the antimelanoma effect 5. Demopoulos, H. B. Effects of low phenylalanine-tyrosine diets on S91 mouse during drug treatment, does not alter median survival compared melanomas. J. Nati. Cancer Inst., 37: 185-190, 1966. 6. Demopoulos, H. B. Effects of reducing the phenylalanine-tyrosine intake of to treated mice maintained on the purified diet. patients with advanced malignant melanoma. Cancer (Phila.), 19: 657-663, In contrast to the complete depletion of asparagine that is 1966.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1982 American Association for Cancer Research. Dietary Influence of Tyrosine and Phenylalanine on the Response of B16 Melanoma to Carbidopa-Levodopa Methyl Ester Chemotherapy

Gary G. Meadows, Herbert F. Pierson, Pokia M. Abdallah, et al.

Cancer Res 1982;42:3056-3063.

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