Download Pteridine and Riboflavin in Tumor Tissue and the Effect

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