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292 N. K O K O L I S , N. M Y L O N A S , AND I. Z I E G L E R Pteridine and Riboflavin in Tumor Tissue and the Effect of Chloramphenicol and Isoxanthopterin \ N . KOKOLIS, N . M Y L O N A S , a n d I. ZIEGLER Department of General B i o l o g y , University of Athens, Botanisches Institut der Technischen Universität, München, and Forschungsgruppe für Biochemie der Gesellschaft für Strahlenund Umweltforschung m.b.H. (Z. Naturforsch. 27 b, 292—295 [1972] ; recived May 5, 1971, revised November 27, 1971) Human squamous cell c a r c i n o m a has elevated levels of tetrahydrobiopterin. A s the level of riboflavin is low, the ratio tetrahydrobiopterin/riboflavin shows values of 5 — 8.3. In contrast, in differentiated tissues with high metabolic activity but low mitotic rate, like submaxillary glands, elevated levels of tetrahydrobiopterin are a c c o m p a n i e d by high content of riboflavin. T h u s the ratio tetrahydrobiopterin/riboflavin in kept as low as about 0.5. Chloramphenicol and, in particular, isoxanthopterin r e d u c e tumor growth in rats and prevent tetrahydrobiopterin accumulation as well. In the foregoing paper 1 a close connection between the formation of a regeneration blastema and a high ratio of tetrahydrobiopterin/isoxanthopterin (TH/X) and/or tetrahydrobiopterin/riboflavin (TH/RB), reaching levels of 3 — 5, has been shown. As a further example of a neoplastic tissue with high mitotic activity tumor tissues will be examined here. Tetrahydrobiopterin, the cofactor in hydroxylation of phenylalanine ( see 1. c. 2 ) has been shown to be present at relatively high concentrations in tissues with intensive protein synthesis. For instance in liver, kidney or spleen it is found at a level of 8.1, 3.0 and 1.2 jug/g fresh weight respectively; in contrast, brain or lung show amounts of only 0.2 — 0.9 jug/g fresh weight 3 ' 4 . However, due to the relatively high concentration of riboflavin 5 ' 6 , TH/RB values of 0.32 in liver, 0.30 in kidney and 1.2 in spleen result. As a further example of a tissue with high protein synthesis submaxillary glands will be examined here and be directly compared with human skin tumors. Both chloramphenicol and isoxanthopterin were found to inhibit the formation of regeneration blastema and to reduce the T H / I X and TH/RB ratios in Triturus cristatus1. Due to its inhibitory effect on ribosomal protein synthesis (see I . e . 7 ) , chloramphenicol has been studied frequently with respect to its action on carcinogenesis. The results were nonuniform. Some authors find little or no influence on tumor growth (e. g. 1. c. 8 ' 9 ) , others an Requests for reprints should b e sent to Frau Dr. I. ZIEGLER, T . U . München, Institut für Botanik, D-8000 München 2, Arcisstr. 21. inhibitory action due to its competition with the cancerogenic drug like N-2-fluorenyldiacetamide (e. g. 1. c. 10> n ) or due to the inhibition of protein synthesis, respiration and DNA synthesis in mouse ascites tumor cells (e. g. 1. c . 1 2 ) . Among unconjugated pteridines, 2,4-diamino6,7-dimethylpteridine 8 for instance acts inhibitively in induced mammary carcinoma. The mode of action is unknown. The marked decrease in T H / I X and TH/RB ratios due to chloramphenicol and isoxanthopterin application, going along with inhibition of regenerative ability, caused us to check both drugs with respect to their action on tumor growth and the appearance of tetrahydrobiopterin. Materials and Methods The submaxillary glands were from young (<3 (3 and $ ? ) rats (Wistar strain). The tumor samples represented human squamous cell carcinoma; they showed marked atypies and numerous mitoses *. Both kinds of tissues were immediately freeze dried after dissection. For the injection experiments, Wistar strain rats weighing between 200 — 250 g ( $ <3 and $ $ ) were used. The cancer cells of type carcinoma T-8 Guerin were suspended in 0,9% NaCl-solution, filtered through a common paper filter and injected subcutaneously into the thigh. The number of injected cells was 80 x 106 per animal. At the end of their lives, the control animals showed tumors at the thigh with a size of 5 x 4 x 4 cm. Injections with isoxanthopterin were made at a dose of 3 /A,g/g body weight at 12 hourly intervals for 10 days; with chloramphenicol at a dose of 200 //g/g body * W e thank the "Institute for Cancer R e s e a r c h " , f o r the material. Unauthenticated Download Date | 8/4/17 1:21 AM Athens, PTERIDINE AND RIBOFLAVIN weight at 6 hourly intervals for 10 days. Both types of injections were made intraperitoneally. For the time table of combined injections of carcinoma cells and drugs see the legend of the figures. Extraction and chromatography were done as described in 1. c. To remove the lipids, the dessicated extracts, however, were treated with 7 ml H 2 0 + 37 ml chloroform ( 2 : 1 ; v/v) containing 0.25% mercaptoethanol + NH4OH (up to pH 10) before further treatment. Qualitative and quantitative determinations of tetrahydrobiopterin and riboflavin were done as described in 1. c. 1 . 1. The characteristics of pteridine and riboflavin pattern in tumor tissue In the submaxillary glands, each of which had a fresh weight of 120 mg, 0.760 jug riboflavin and 0.330 jug tetrahydrobiopterin were present. The determinations, which were made at least 6 fold, showed only minimal variations ( < 3 % ) . The human skin tumors avaraged a fresh weight of 1.5 g and contained hydrobiopterin and 1.490 — 2.530 /ug tetra- 0.270 — 0.330 jug riboflavin. Isoxanthopterin was not present in either type of tissue. Submaxillary g l a n d pg/g fresh weight Tetrahydrobiopterin 2,75 Riboflavin 6,3 Ratio Tetrahydrobiopterin: Riboflavin 0,44 s q u a m o u s cell c a r c i n o m a pg/g fresh weight 1 — 1,65 0,18—0,22 5—8,3 Table 1. Tetrahydrobiopterin and riboflavin in submaxillar)' glands of rats and in human squamous cell carcinoma. Table 1 shows the absolute values for tetrahydrobiopterin and riboflavin in 1 g fresh weight of tissue and the resulting ratios of TH/RB. It demonstrates that the tumor samples have a much higher ratio, which is partially because of their elevated level of tetrahydrobiopterin, but mostly due to the fact that riboflavin is not accumulated in the tumor sample. TUMOR 293 TISSUE 2. The action of chloramphenicol and isoxanthopterin on the growth and the tetrahydrobiopterin content of transplanted tumor tissue a) Chloramphenicol As seen in Fig. 1 a, animals into which carcinoma cells were transplanted showed earlier tumor growth than those which received chloramphenicol for 10 days starting with the day of tumor transplantation. Moreover their death was much more delayed. b) Results IN Isoxanthopterin Fig. 1 b demonstrates the action of isoxanthopterin which was injected for 10 days, starting also with the day of application of tumor cells. The delay in tumor growth and the increase in life time are even more marked than after injection of chloramphenicol. As in squamous cell carcinoma tetrahydrobiopterin was found to be accumulated in the transplanted growing tumors, even no quantitative determinations were made. It could not be detected at all in muscle and dermal tissue of normal animals and those in which tumor development was prevented by both drugs. Discussion The data given above and earlier results 1 show that tumor tissue and regeneration blastema are characterized by high T H / I X and/or TH/RB ratios. Both neoplastic tissues are characterized by high mitotic activity and ribosomal protein synthesis; moreover, in contrast to other tissues with high metabolic activity alone, like liver, they are not fully differentiated. The same is true for larval skin before metamorphosis, which also is characterized by high T H / I X and TH/RB ratios 1 3 . Drugs inhibiting or promoting these growth characteristics prove to affect both ratios in a parallel way, indicating a very close connection. With respect to the interdependence of both characteristics for tumor tissues, the same are present in the growing regeneration bud and were discussed in 1. c. In the case of hepatoma and other malignant neoplasms, xanthinoxidase, which catabolizes tetrahydrobiopterin 14 , already has been Unauthenticated Download Date | 8/4/17 1:21 AM 294 days N. K O K O L I S , 0 2 4 6 8 N. M Y L O N A S , AND 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 I—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—r I. Z I E G L E R Fig. 1 a. Tumor growth (carcinoma T-8 Guerin) and survival of rats without and with injection of chloramphenicol. days 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 I—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i i i i i i i i i i i r Fig. 1 b. Tumor growth (carcinoma T-8 Guerin) and survival of rats without and with inection of isoxanthopterin. tumor transplantation and simultaneous beginning, -j- of drug application hi'iiiumi duration of drug application, = mals without palpable tumor, animals with palpable tumor, J spontaneous death. investigated. These tissues show reduced xanthinoxidase activity 15 . In consequence, xanthinoxidase, injected into mice, bearing spontaneous mammary tumors, shows antitumor effects 16 . One may assume that decreased xanthinoxidase activity, resulting in ^ timing = ani- low isoxanthopterin and riboflavin levels but keeping the amount of tetrahydrobiopterin high (see I . e . 1 ) , is directly involved here. Thus the regulation of this enzyme may be a central point in neoplastic growth. Unauthenticated Download Date | 8/4/17 1:21 AM PTERIDINE AND RIBOFLAVIN IN TUMOR TISSUE One of us (N. K.) is indebted to Stiftung for a grant and generous help. Humboldt- 9 10 2 S. KAUFMAN, A n n . 3 H. REMBOLD, Pteridine Chemistry, Pergamon Press, Oxford 1964. 12 H . REMBOLD a n d 13 4 5 6 7 Naturforsch. 11 [1972], H. 36, 171 CH. STOCK, METZGER, Z. Naturforsch. 2 2 b , 827 [1967]. Biochemist's H a n d b o o k , Ed. C. LONG, E. U. F. N. SON, L o n d o n 1961. HOPPE-SEYLER/THIERFELDER, Handb. der physiol.- u. path.chem. Analyse, B d . I I I / 2 . Springer-Verlag, Berlin-Heidelberg-New Y o r k 1955. R . SCHWEET a n d R . HEINTZ, A n n . R e v . B i o c h e m . 3 5 , A . P . D A V I S , M . G R U E N S T E I N , a n d M . B . SHIMKIN, H. CH. REILLY, a n d 723 M. [Paris] 54, [1967]. J . H . WEISBERGER, Y . SHIRASU, P . H . G R A N T H A M , a n d D . HALDAR 1009 M. [1958]. K . WEISBURGER, J . b i o l . C h e m i s t r y 2 4 2 , 3 7 2 [1967]. and K. E. [1967]. B . FREEMAN, C a n a d . J . B i o c h e m . 46, [1968]. N . KOKOLIS and I. ZIEGLER, Z. Naturforsch. 23 b, 860 [1968]. 14 H. REMBOLD, Chemistry and B i o l o g y of Pteridines, Int. A c a d . Printing Co., T o k y o 1970. 15 P H . FEIGELSON, J . E . U L T M A N , S . H A R R I S , a n d T H . DASH- MAN, Cancer Research 1 9 , 1 2 3 0 [ 1 9 5 9 ] . 10 A . H A D D O W , E . E . H O R N I N G , F . B E R G E L , G . M . TIMMIS, T . OSDENE, T . S. BRAY, R. C. AVIS, and A. BROWN, Brit. Emp. Cancer Camp. Ann. Rept. 3 1 , 35 [ 1 9 5 3 ] . [1966], 8 C. A . LACASSAGNE and L. HURST, Bull. Cancer 405 N . K O K O L I S , N . M Y L O N A S , a n d I . ZIEGLER, Z . Rev. Biodiem. SUGIURA, SCHMID, C a n c e r R e s . 1 8 , 6 6 1 27 b, 285 K. 295 Cancer Res., Chemotherapy 26, 1 [ 1 9 6 6 ] . Unauthenticated Download Date | 8/4/17 1:21 AM