Download Quantitative Biochemical Differences between

Quantitative Biochemical Differences between
Tumor and Host Tissue
VI. 6-Aminonicotinamide Antagonism of DPN-dependent
Enzymatic Systems*
L. S. DIETRICH,tLEATRICEA. KAPLAN,IRAM. FRIEDLAND,!
ANDDANIELS. MARTINA
(Departments of Biochemistry and Surgery, Collegeof Physicians and Surgeons, Columbia University, New York 32, N.Y.)
The general mechanism whereby a tumor, an
integral part of the host, may be selectively de
stroyed without permanently altering the other
tissues of the host is unknown. Extensive research
has been directed toward finding something unique
in tumor metabolism. These studies indicate that
the same metabolic pathways and metabolites
found in tumor tissue are also found in other host
tissues; i.e., the metabolism of neoplasms does not
differ qualitatively from other animal tissues (18,
20, 34, 35). There are, however, many reports that
chemical compounds, alone or in combination, can
destroy or alter the growth rate of experimental
neoplasms without producing overt toxicity to the
host. Thus, there must be something different bio
chemically between neoplasms and other tissues.
Logically, if these biochemical differences are not
qualitative in nature, they must be quantitative.
Many metabolites, co-factors, and enzymes have
been reported to be in lower concentration in host
neoplasms than in the majority of the normal host
tissues (2, 3, 10, 11, 14, 17, 20, 22, 27-29, 33-35).
From these observations, together with a more ma
ture understanding of the mechanism of antimetabolite action, had evolved a working hypothe
sis that metabolic pathways in malignant tissues
can be blocked while producing minimal effects on
the same systems, which are more abundant in
normal tissues (1, 4, 21-23, 26-29).
For the past several years we have attempted to
test the validity of this hypothesis employing bio
logical and biochemical studies (7, 21-29). To date
these studies, although very suggestive, do not
conclusively demonstrate that the biological re
sults observed were actually the result of an enzy
matic or co-factor differential between non-neoplastic and neoplastic cells. However, few of the
compounds available were powerful antimetabolites capable of producing effects lasting long
enough to be measured biochemically, and even in
those cases of demonstrable enzymatic alterations
(7, 26, 27) the systems affected could not be classi
fied as vital for cellular life.
Recently, we obtained a powerful niacin antago
nist, 6-aminonicotinamide (6-AN) (13). This com
pound is capable of causing the 755 tumor grown
in C57 mice to regress (23). Administration of 6AN markedly inhibits the activities of various
pyridine nucleotide (PN)-dependent enzymes, in
particular those systems associated with mitochondrial oxidative phosphorylation processes (5,
9). Systems of this type fall indisputably into the
category of enzymes essential for cellular life.
The potency of this compound at both the bio
logical and biochemical level suggested that 6-AN
* Supported by grants-in-aid from the Williams-Waterman
might serve as an important tool to determine
Fund, the National Vitamin Foundation, and the United
States Public Health Service (CY-2446 [C2] CY).
whether quantitative biochemical differences ex
plain why certain tissue, e.g., tumor, is destroyed
t Present address: Department of Biochemistry, University
of Miami, School of Medicine, Coral Gables 34, Florida.
and other tissues, e.g., normal host tissues, are
ÃŽ
Present address: Department of Surgery, University of unaffected by the same level of drug. Studies em
Miami, School of Medicine, Jackson Memorial Hospital,
ploying the mitochondrial DPN-dependent sys
Miami 86, Florida. Formerly Daniel M. Shapiro.
tems dealing with the oxidation of a-ketoglutarate,
The following abbreviations are used: diphosphopyridine
malate,
and /3-hydroxybutyrate, reported in this
nucleotide, DPN; 6-aminonicotinamide analog of DPN, 6amino-DPN; 6-aminonicotinamide, 6-AN; adenosine mono-, communication, indicate that quantitative
bio
di-, tri-phosphate, AMP, ADP, and ATP, respectively.
chemical differences may be utilized to produce
selective tissue toxicity.
Received for publication June 16, 1958.
1272
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1958 American Association for Cancer Research.
DIETRICH et al.—6-Aminonicptinamide
and Enzyme Systems
1273
redissolved in the desired volume of 60 per cent ethanol. These
MATERIALS AND METHODS
conditions quantitatively hydrolyze the nicotinamide-ribose
General.—C57BLmice, 2-4 months old, weighing 18-22
bond of DPN and TPN without affecting the 6-AN-ribose
gm., were housed in plastic cages in an air-conditioned, con
linkage. Negligible hydrolysis of pyrophosphate bonds was ob
stant-temperature room (74°
F.). All mice received, ad libitum,
served.2 The crude nucleotides were then separated chromatoa diet of Rockland pellets and water. The neoplasm employed
graphically (5). Where the 6-amino-DPN areas were heavily
was the mammary adenoearcinoma 755, transplanted into the
axillary region by the usual trocar technic. 6-AN dissolved in contaminated with AMP, all material moving more slowly than
6-amino-DPN was cut away, and the sheets were redeveloped
saline was injected intraperitoneally.
Enzymatic studies.—Theanimals were sacrificed by cervical in isopropanol :glacial acetic acidiwater (60:30:10) (15). This
rupture 24 hours after the cessation of therapy. The tissues system moves bases, nucleosides, and mononucleotides, but
not dinucleotides and polyphosphate nucleotides. With this
were immediately removed, chilled in crushed ice, blotted dry,
and homogenized in 0.25 M sucrose containing 0.02 M nico- technic the contaminating AMP was removed from the areas
occupied by 6-amino-DPN. The papers were then washed chroInumide and 0.004 M versene in a glass homogenizer of the matographically in saturated N-butanol-NHiHCOs atmos
Potter type (19).
The capacity of tissue homogenates to convert /3-hydroxy- phere, dried, and the areas occupied by 6-amino-DPN were cut
out and concentrated into a small area (4—6
sq. cm.) of paper by
butyrate to acetoacetate was measured as follows: MgCU,
ascending chromatography with 0.1 M acetate (pH 4.5). The
0.01 M; ATP, 0.0014 M; nicotinamide, 0.06 M; potassium phos
areas containing the concentrated 6-amino-DPN were cut into
phate buffer (pH 7.4), 0.015 M;and the appropria te volume of tis
small
pieces and eluted overnight in 2 ml. of 0.1 M HC1. Blank
sue homogenate was added to the ice-cold reaction vessels. Cold
sheets of paper were treated similarly. Spectra were determined
isotonic KC1 was added to give, after the addition of substrate,
against the similarly treated blanks with a Beckman DU
a final volume of 2 ml. Immediately before the vessels were spectrophotometer.
The molar extinction at E30j of 6.4 X 10'
placed into a constant-temperature water bath, 0.2 ml. of a
was
used
in
calculating
the amount of 6-amino-DPN present.
0.2 M |8-hydroxybutyrate solution was added. All reactants
The procedure employed in the isolation and analysis of
were neutralized with KOH prior to addition. Incubation was
carried out at 37°C. with agitation for 80 minutes, at which AMP, ADP, and ATP is the same as reported elsewhere (5).
tune the reaction was stopped by the addition of 0.5 ml. of 15
RESULTS
per cent TCA. Acetoacetate analyses (32) were carried out on
The normal activities of the enzymatic systems
the supernatant material obtained upon centrifugation. Tis
sues, as homogenates. were added as follows: liver, 20 mg.; kid
chosen as models were determined in the 755 tu
ney, 10 mg.; brain, 30 mg.; lung and tumor, 200 mg.; and skel
mor and six normal tissues of the C57BL mouse
etal muscle, 300 mg.
(Tables 1-3). The capacity to convert a-ketoThe ability of tissue homogenates to convert a-ketoglutarate to citrate was measured as follows: pyruvate, 0.011 M; glutarate and malate to citrate and /3-hydroxyATP, 0.007 M; MgClj, 0.008 M; nicotinamide, 0.04 M; potas
butyrate to acetoacetate was found to be highest
sium phosphate buffer (pH 7.4), 0.001 M; fluorocitrate, 2.15 X
10~4M;and the appropriate volume of tissue homogenate was in liver. The other tissues had the following order
added to ice-cold reaction vessels. Ice-cold isotonic KC1 was of decreasing activities: kidney, brain, heart, 755
added to give, after the addition of substrate, a final volume of tumor, skeletal muscle, and lung. The relative ac
tivities of all three systems paralleled each other in
2.7 ml. Immediately before the vessels were placed in a con
stant-temperature water bath, 0.2 ml. of a 0.10 M a-ketogluall tissues.
tarate solution was added. All reactants were neutralized with
6-AN was converted, in vivo,to 6-AN analogs of
KOH prior to addition. Incubation was carried out at 37°C.
DPN
and TPN (5, 9). These compounds under
with agitation for 30 minutes, at which time the reaction was
stopped by the addition of 0.5 ml. of 30 trichloroacetic acid went none of the addition reactions typical of nat
(TCA). Citrate analyses (16) were carried out on the super
ural pyridine nucleotides. It has been postulated
natant material obtained upon centrifugation. Tissues, as that these impotent pyridine nucleotides become
homogenates, were added as follows: kidney and liver, 50 mg.; bound, in vivo, to available apo-dehydrogenases,
brain and heart, 100 mg. ; and tumor, lung, and skeletal muscle,
producing inactive holo-enzymes (5,9). Within the
200 mg.
The capacity of tissue homogenates to convert malate to cell these unnatural pyridine nucleotides must
citrate was determined in a manner identical to that used in the compete with the normal pyridine nucleo tide pool
o-ketoglutarate studies, except that the substrate used was for the apo-dehydrogenases. Thus, the level of
malate, 0.008 M. In the malate studies, tissue homogenates
were added as follows: kidney and liver, 15 mg.; brain, 25 mg.; DPN and DPNH present in the cell.is a controlling
heart and tumor, 100 mg.; lung and skeletal muscle, 200 mg. factor in regard to the ability of 6-AN to antago
nize the DPN-dependent enzymes of the cell. The
The tissue concentrations of DPN and DPNH were deter
mined enzymatically according to the procedure of Jederkin
1The studies reported in this paper are all based on whole tis
and Weinhouse (12).
Nucleotide studies.—Micereceived a single injection of 6- sues. Therefore, any effect on minority cell types within these
AN (200 mg/kg) 14-21 days after implantation. The animals tissues would go unobserved owing to dilution by the major
were decapitated 18 hours later, the tissues1 immediately re
cell types. This could explain the anomaly concerning the effect
moved and dropped into ice-cold 3 per cent perchloric acid. of 6-AN on the anterior horn cells and brain stem nuclei (30)
and the enzymatic impotency of 6-AN, at the therapeutic lev
Sufficient 3 per cent perchloric acid was added to give 9 vol
umes, and the tissues were homogenized and centrifuged in the els on whole brain tissue. It is possible that the tissues of the
cold. The acid-soluble nucleotides were adsorbed on acid- spinal cord and brain stem either have lower DPN-dependent
enzymatic capacities or can utilize 6-AN more efficiently than
washed Nuchar and eluted with ammoniacal aqueous ethanol
(5, 31). The ammoniacal eluate was allowed to stand at room the other tissues of the central nervous system.
temperature for 24 hours, concentrated in vacuo at 40°C., and
2L. S. Dietrich and I. M. Friedland, unpublished data.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1958 American Association for Cancer Research.
Cancer Research
1274
Vol. 18, December,
1958
lowest in co-factor concentration and enzymatic
activity. The 755 tumor, lung, and skeletal muscle
had the lowest activities of the three systems stud
ied and also fell into the lowest magnitude in re
gard to DPN concentration. These tissues should
be antagonized at the lowest level of 6-AN effec
tive against the tumor. Increasing amounts of drug
administered should antagonize the DPN-dependent enzymes in the other tissues exhibiting higher
enzymatic activities and DPN concentrations.
Brain and heart tissue should be the most suscep
tible to inhibition after lung, tumor, and skeletal
muscle. Heart could conceivably be more resistant
to 6-AN than brain, owing to the fact that it con
tains DPN levels comparable to liver and kidney.
DPN and DPNH levels of the tissues studied
(Table 4) were in the following order of decreasing
concentration: liver, kidney, heart, brain, skeletal
muscle, lung, and tumor. These values fell into
two separate magnitudes with heart, liver, and
kidney having 2-3 times the concentration of total
DPN as does skeletal muscle, lung, brain, and the
755 tumor.
Our working hypothesis states that selective
toxicity of tissues is, primarily, due to quantitative
biochemical differences between tissues, and that
tissues with the lowest co-factor concentration and
lowest enzyme activities are the most vulnerable.
If this is the case, 6-AN administration at the
therapeutic level should antagonize those tissues
TABLE 1
THEEFFECTOFO-AMINONICOTINAMIDE
ADMINISTRATION
ONTHECONVERSION
OF/J-HYDHOXYBDTYRATE
TOACETOACETATE
INMOUSE
TISSUES
AND755TUMOE*
ENZYMATIC
ACTIVITY±S.E.
(AE450/30
TISSUE
LiverKidneyBrainHeartTumor
TreatedControl
mgAgX4
202 + 11 (10)
152
±12(10)74
+ 8 (9)
(lO)112
122±6
± 2(10)
68± 1(10)16
8(8)
2(8)6.1±0.4(7)
87±
TreatedControl
+ 3 (7)
96
2(7)22
+
+ 6 (8)
±2(8)0.22
19
TreatedControl
(755)LungSkeletal TreatedControl
mgAgX4146
+ 17(9)
(9)6.2±0.5
254
±15
TreatedControl
TreatedControl
MINUTES/GM TISSUE WET WT)
mgAgX4
316 + 10(8)
7(8)248
227±
mgAgXS
270 ±12 (8)
280±
9(8)85±
(8)
1.9±0.4(s)1«
3.4
0.2(10)2.2
+
+ 0.1 (21)
0.7 + 0.2(21)8
muscleControl Treated*
* Dosage is mg/kg X no. days administered.
analyzed. S.E. = Standard Error.
+ 0.02(1
0.22±0.02('
The figures in parentheses
represent
the number of animals
TABLE 2
THEEFFECTOFB-AMINONICOTINAMIDE
ADMINISTRATION
ONTHEABILITY
OFMOUSE
TISSUEAND755TUMOR
To CONVERT
O-KETOGLUTAHATE
TOCITRATE*
/¿MOLESCITRATE±S.E. PHODUCED/30 UIN/GH TISSUE WET WT
TISSUE
Liver
2 mgAgXS
Control
Treated
Kidney
Control
Treated
Brain
Control
Treated
Heart
Control
Treated
Tumor
(755)
Skeletal
muscle
Lung
Control
Treated
Control
Treated
8 mgAgX4
25.8±2.4(6)
23.3±1.0 (6)
18 mgAgX4
29.7 + 0.9(7)
23.7±2.1 (7)
25.7±0.7(9)
23.0±1.2(9)
12.8±0.6 (6)
11.8 + 0.4 (6)
9.8 + 0.6 (7)
7.4±0.2(7)
10.3 + 0.8 (6)
10.7 + 0.4(8)
3.5 + 0.4(10)
1.1±0.1 (lO)
3.7 + 0.2(10)
1.9±0.2(10)
2.9 + 0.1 (7)
2.3±0.3(7)
2.0 + 0.08(10)
Control
1.6±0.06 (lO)
Treated
* Dosage is mg/kg X no. days administered. Figures in parentheses
animals. S.E. = Standard Error.
represent
2.9 + 0.1 (7)
2.9 + 0.1 (7)
the number
of
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1958 American Association for Cancer Research.
DIETRICH et al.—6-Aminonicotinamide
Liver and kidney tissue, if antagonized, should re
quire higher levels of 6-AN than the other tissues
studied.
To test this hypothesis, tumor-bearing mice re
ceived 6-AN at various dosages. The lowest level
given was the standard therapeutic dose used pre
viously (23), 2 mg/kg once a day for 8 days. Pro
gressively higher dosages of 8 mg/kg for 4 days, 12
mg/kg for 4 days, and 16 mg/kg for 4 days were
employed. Higher dosage regimens were too toxic
for study. Enzymatic analyses of the tissues of
these animals were carried out and compared with
those for similar animals that were untreated. The
effects of these various levels of 6-AN on the abil
ity of homogenates of normal mouse tissues and
and Enzyme Systems
1275
lower level of 6-AN. Administration of 6-AN at a
level of 8 mg/kg for 4 days markedly increased the
inhibition of the oxidation of /3-hydroxybutyrate
in tumor tissue. This level of drug had no observed
effect on liver and kidney tissue. At a level of 12
mg/kg for 4 days, significant inhibition of liver
tissue was observed along with a probable inhibi
tion in brain. At a level of 16 mg/kg a definite in
hibition of the oxidation of /3-hydroxybutyrate
was observed in brain tissue. The inhibition ob
served in kidney at this level of 6-AN is question
able. This high, extremely toxic level of 6-AN had
no effect on cardiac or skeletal muscle.
Similar results were observed in the studies on
oxidation of a-ketoglutarate to citrate (Tables 2
TABLE3
THE EFFECTOFÔ-AMINONICOTINAMIDE
ADMINISTRATION
ONTHE CONVERSION
OFMALATE
TOCITRATEIN MOUSETISSUEAND755 TUMORHOMOGENATES*
,, M.I 1.1s CITHATE±S.E.
TISSUE
Liver
PBODDCED/SO MIN/GM TISSUE WET WT
12 mgAgX4
86 + 8(10)
Control
Treated
74±5(10)
Kidney
Control
Treated
Brain
57 ±1(8)
Control
58 + 1 (8)
Treated
Heart
Control
Treated
Tumor
13.7 + 0.9 (10)
Control
11.4 + 0.5(10)
Treated
Skeletal
Control
muscle
Treated
Lung
2.910.2 (10)
Control
3.2 + 0.2(9)
Treated
* Dosage is mg/kg X no. days administered. Figures in parentheses represent the number
lyzed. S.E. = Standard Error.
»mg/kg X8
the 755 tumor to convert /J-hydroxybutyrate to
acetoacetate (Table 1), a-ketoglutarate and malate
to citrate (Tables 2 and 3, respectively) are com
pared.
A statistical evaluation of these three tables
(Tables 1-3) is presented in Table 5. Here the "t"
values, indicating the significance of the difference
between the control and 6-AN-treated activities,
are recorded. We have considered the results to be
beyond chance variation when "t" is 3.0 or higher.
From these data it can be seen that 6-AN, at a
level of 2 mg/kg for 8 days, produced a marked
inhibition of j3-hydroxybutyrate oxidation in tu
mor and lung tissue but not in skeletal muscle
(Tables 1 and 5). The same level of drug had no
observed effect on brain and liver tissue. The other
tissues were not analyzed at this level, but, since
the oxidation of ß-hydroxybutyrate was unaltered
in these tissues by higher levels of 6-AN, we may
assume that the enzyme was uninhibited by the
8 mgAgX4
85 + 6(8)
91 + 13 (8)
116+ 6 (9)
121±4(9)
16 mg/kgX4
82 ±3(10)
77 + 4 (9)
113+ 4(9)
118+ 5 (10)
48 + 2(10)
45 + 1 (10)
15.5 + 1.3(8)
13.6 + 0.4 (8)
14.0±1.2(10)
9.7 + 0.3(10)
5.5±0.4(7)
4.4 + 0.5(7)
3.6 + 0.3 (8)
2.9 + 0.5(8)
of animals ana
TABLE4
CONCENTRATIONS
OFDPN ANDDPNHIN VARIOUS
TISSUES OF THE C57 MOUSE*
TissueLiverKidneyHeartBrainSkeletal
15(9)389
+
(11)244
+ 10
(10)339
+ 14
(10)277
+ 22
(12)208+
+ 13
12(10)88+
+
4(10)268+
3(10)21+
7(12)100+
8(10)135+
muscleLung755
(11)137+
5
3(10)22+
2(8)Total(iig/gm)708633616296289235159
9(8)DPNHG«g/gm±S.E.)227
TumorDPNGig/gm±S.E.)481
* Figures in parentheses represent the no. of analyses run.
Each analysis was from individual tissues in the case of liver,
tumor, and skeletal muscle. Tissues from two animals were
pooled in the case of kidney, heart, brain, and lung.
S.E. = standard error.
and 5). Under these conditions, this system did not
seem to be as sensitive to 6-AN as the ß-hydroxybutyrate system. 6-AN at a level of 2 mg/kg for a
period of 8 days significantly inhibited the oxida
tion of o-ketoglutarate in tumor tissue and lung
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1958 American Association for Cancer Research.
Cancer Research
1276
tissue. Increasing the 6-AN level to 8 mg/kg for 4
days had no effect on the oxidation of alpha-ketoglutarate in skeletal muscle, brain, or liver. Rais
ing the dose of 6-AN to 16 mg/kg for 4 days sig
nificantly inhibited the oxidation of a-ketoglutarate in brain and had a possible effect on liver.
Kidney, skeletal muscle, and heart activities were
unaltered by this level of 6-AN.
Vol. 18, December,
1958
The oxidation of malate to citrate (Tables 3 and
5) was very resistant to antagonism by 6-AN. The
only tissue in which a significant inhibition of the
oxidation of malate was observed was in the case
of the 755 tumor and even in this case only at the
very toxic level of 16 mg/kg for 4 days.
The concentration of 6-amino-DPN in the 755
tumor and six normal tissues is reported in Table
TABLE 5
STATISTICAL
EVALUATION
OF THE EFFECTOF VARIOUS
DOSAGES
OF 6-AN ON
MOUSE
TISSUES
The ability to convert /3-hydroxybutyrate to acetoacetate and a-ketoglutarate
and malate to citrate was tested*
DOSAQE(mg/kgXday
|3-Hydroxybutyrate
2X8
8X4
12X4
Liver
Kidney
Brain
0.7
1.3
0.4
3.1
Heart
Tumor
a-Ketoglutarate
Malate
inj.)
TI88DE
Lung
Skeletal muscle
Liver
Kidney
Brain
Heart
Tumor
Lung
Skeletal muscle
Liver
Kidney
Brain
Heart
Tumor
Skeletal muscle
Lung
* Values are expressed "t" values, when "t" -
2.6
8.8
18X4
2.4
4.5
0.5
6.6
7.8
1.0
2.6
1.9
3.8
1.0
1.0
1.4
6.8
4.0
8.3
1.7
1.0
1.0
1.0
1.0
1.0
2.8
1.0
mi —¿
1.0
1.0
1.5
1.6
3.3
1.0
1.3
, mi = control activities,
and m2 = 6-AN-treated activities.
The results are consistent beyond chance variation when "t" is higher than S.O.
6. The 755 tumor, kidney, and liver were found to
have the highest concentration of the analog fol
lowed by brain, lung, heart, and skeletal muscle.
With the ratio of total DPN :6-amino-DPN
8-Amino-DPNt
Total DPN/
(Table 6) used as a guide, it can be seen that the
Tissue
(/ig/gm)93.075.0100.052.024.322.06.36-Amino-DPN7102602840
tissues studied fell roughly into three different
Kidney
magnitudes. Tumor had the lowest ratio, and
Liver
skeletal and cardiac muscle had the highest. The
755 tumor
intermediate tissues—liver, kidney, brain, and
Brain
Lung
lung—had similar DPN :6-amino-DPN ratios.
Heart
The effect of 6-AN administration on the natu
Skeletal muscle
* 6-AN was administered at a level of 200 mg/kg
ral adenine nucleotides AMP, ADP, and ATP pres
18 hours before sacrifice. Values are the average of
ent in lung and tumor tissue is presented in Chart
duplicate analyses from ten to fifteen pooled tissues
1. These data are from animals receiving 6-AN at a
in the case of liver and tumor and 60-80 pooled tis
sues for kidney, brain, heart, and skeletal muscle. See
level of 8 mg/kg for 4 days. The same effect has
"Materials and Methods" for details.
been observed at the 2 mg/kg therapeutic dose.2
t Values calculated on the basis of I-'.•¿..,,::>
Mof the
The administration of 6-AN had no effect on the
original perchloric acid filtrates.
Previous work has shown that at this or other
nucleotide pattern of lung tissue. Tumor tissue,
levels of 6-AN no significant changes in the DPN
concentrations of the whole tissues are observed.*
as previously reported (5), was markedly affected;
TABLE 6
EFFECTIF Ô-AMINONICOTINAMIDE
ONTHECON
CENTRATION
OF6-AMINO-DPN
IN MOUSETISSUES*
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1958 American Association for Cancer Research.
DIETRICH et al.—6-Aminonicotinamide
the AMP levels were elevated threefold, and the
5'-polyphosphate nucleotides, ATP in particular,
were greatly decreased.
DISCUSSION
The oxidation of /3-hydroxybutyrate is the most
sensitive to 6-AN antagonism of the systems stud
ied. The oxidation of a-ketoglutarate is also read
ily antagonized by 6-AN. Malate oxidation is, on
the other hand, only slightly antagonized by this
anti-metabolite. Studies regarding the effect of
6-AN administration on other DPN-dependent
enzymatic systems show that 3-phosphoglyceraldehyde dehydrogenase (5, 9) is markedly an-
and Enzyme Systems
1277
dose of the ribo flavin analog, flavo tin (7), mark
edly inhibited the xanthine oxidase activity of 755
tumor but had little effect on the xanthine oxidase
activity of the livers from the same animals. The
livers had 7-8 times the xanthine oxidase activity
of the tumors. These data follow the hypothesis
first presented by Ackermann and Potter (1)
". . . it should be possible to inhibit an enzyme
present in cancer tissue in small amounts, while
producing only partial inactivation of the enzyme
in tissues containing larger amounts."
The present study shows that the niacin antago
nist 6-AN antagonizes various DPN-dependent
enzymes in a similar manner. At a therapeutic dose
Control
6-AN Treated
(8mg/Kg/day
for 4 days)
ATP
ADP
755
AMP
TUMOR
ATP
ADP
AMP
LUNG
CHART1.—Effect of 6-aminonicotinamide administration
on the acid-soluble nucleotides of mouse tissue.
Tumor values are the average of three separate experiments
consisting of ten to fifteen pooled tumors per experiment. The
lung values are an average of two separate experiments con
sisting of 20-40 pooled lungs per experiment.
See "Materials and Methods" for details.
tagonized, while pyruvate2 and lactic dehydro
genase (5, 9) are insensitive to 6-AN. These data
are in keeping with previous studies in regard to
deoxypyridoxine antagonism (6) of transaminase
and pantethine antagonism of co-enzyme A-requiring systems (8) and again emphasize the fact
that metabolic antagonists do not antagonize all
systems dependent on the metabolite to the same
degree.
Previous studies from this laboratory have dem
onstrated that deoxypyridoxine antagonizes glutamic-aspartic transaminase activities of mouse
tissues having low transaminase activities, e.g.,
tumor, testes, and lung, to a far greater degree
than tissues with higher transaminase activities,
e.g., heart, liver, and kidney (27). A therapeutic
of 6-AN (2 mg/kg for 8 days) the oxidation of ßhydroxybutyrate and a-ketoglutarate is antago
nized in tumor and lung tissue. At this level of 6AN administration no inhibition of these systems
was observed in tissues such as liver, brain, and
kidney, which have much higher enzymatic activi
ties. Increasing the level of drug caused increasing
toxicity (as measured by enzymatic inhibition) in
tissues with high concentrations of DPN and high
activities of the DPN-dependent enzymatic sys
tems studied.
Since 6-AN appears to exert its effect by form
ing the enzymatically inactive 6-amino-DPN, one
criterion of its inhibitory capacity would be the
tissue concentration of this pseudo-coenzyme. Pre
cise quantitative determinations of 6-amino-DPN
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1958 American Association for Cancer Research.
1278
Cancer Research
in tissues following 6-AN at the lower dosages used
for our enzymatic studies are not feasible, because
available methods are not sensitive enough to ac
curately measure the small amounts of analog
present. We have assumed that the levels of 6amino-DPN found after massive injections of 6AN (200 mg/kg) parallel those found following the
various levels of 6-AN administration used in the
enzymatic studies. Qualitative estimates of the 6amino-DPN found at these lower dosages confirm
this assumption.2 The data (Table 6) appear to
explain the discrepancies observed following 6-AN
therapy on the oxidation of /3-hydroxybutyrate
and a-ketoglutarate (Tables 1 and 2). For example,
skeletal muscle, which has the same DPN level
and enzymatic capacity as lung and tumor to
oxidize these two metabolites, was unaffected by
6-AN therapy. These latter two tissues were, how
ever, markedly inhibited by the lowest level of 6AN (Tables 1 and 2). However, very little 6amino-DPN could be demonstrated in skeletal
muscle after a massive dose of 6-AN, in contrast to
the relatively high amounts found in lung and tu
mor (Table 6). This may be owing to the inability
of 6-AN to efficiently cross the cellular membrane
or the inability of skeletal muscle to synthesize ef
fectively 6-AN nucleotides from 6-AN. To justify
this latter explanation, one would have to postu
late that 6-AN is incorporated into the nucleotide
in a manner different from that whereby nicotinamide is converted into the nucleotide, since DPN
is present in skeletal muscle in amounts compa
rable to that in tumor and lung. Another possibil
ity is that these low levels of 6-AN nucleotides
found in skeletal muscle are merely a reflection of a
slow turnover of nucleotides characteristic of skel
etal muscle. Whatever the mechanism, the pres
ence of low levels of 6-AN nucleotides in skeletal
muscle would explain why no enzymatic antago
nism is observed in this tissue after 6-AN adminis
tration. In cardiac muscle, a similar situation ex
ists. Its high concentration of natural DPN and
the relatively low level of 6-amino-DPN would ex
plain the lack of effect of 6-AN in this tissue.
Tumor tissue which has the same DPN magni
tude as skeletal muscle and brain accumulates 6amino-DPN to the same degree as liver and kid
ney, which have higher levels of the natural cofactor. Thus, besides having a lower enzymatic
capacity of DPN-dependent enzymes, the 755 tu
mor also has the lowest DPN :6-amino-DPN ratio
of any of the tissues studied. The question now
arises whether the vulnerability of the 755 tumor
could be explained solely by this ratio, the low ac
tivities of the DPN-dependent enzymes being
merely coincidental. Certainly the capacity of a
Vol. 18, December,
1958
tissue to utilize an antagonist and the concentra
tion of the normal metabolite in the cell are impor
tant factors in enzymatic inhibition. However,
lung tissue with a DPN :6-amino-DPN ratio simi
lar to brain, liver, and kidney is as sensitive enzymatically to 6-AN as tumor (Tables 1-3). Thus, if
6-AN is utilized by a tissue to a degree sufficient to
produce an effective cofactor: antagonist ratio, the
primary factor affecting enzyme antagonism
would appear to be the enzymatic capacities of the
tissue.
The action of 6-AN on the acid-soluble nucleo
tide pattern, in vivo, suggests that 6-AN appears
to be starving the tumor tissue (Chart 1). The
lowering of the 5'-polyphosphate nucleotides, espe
cially ATP, with a concomitant increase in 5'monophosphate nucleotides, AMP in particular,
presents a theoretical picture of energy depriva
tion. Similar results have been obtained in respir
ing mitochondrial preparations that have been ex
posed to cyanide or dinitrophenol (21). In the case
of 6-AN antagonism, inhibition probably occurs at
the initial step of electron transport in the mito
chondria. 6-AN nucleotide analogs of DPN and
TPN are incapable of undergoing the normal oxi
dation and reduction reactions characteristic of
the natural pyridine nucleotides (5). These 6-AN
nucleotides have been postulated to become bound
to mitochondrial pyridine nucleotide-dependent
dehydrogenases (5). Once such an analog-enzyme
complex is formed, normal dehydrogenation would
stop, and no electrons would enter the cytochrome
system. In this case no oxygen would be con
sumed, and no oxidative phosphorylation could
occur. This impaired ATP synthesis would then
limit vital endergonic biochemical reactions, e.g.,
growth.
The 755 tumor, like other neoplasms, has a low
capacity of the respiratory enzymes involved in
oxidative phosphorylation (5). This is coupled
with a high requirement for the product of oxida
tive phosphorylation, ATP, as surmised by its
rapid growth. The data (Chart 1) indicate that the
755 tumor, when inhibited by 6-AN, is consuming
ATP faster than it is resynthesized. It is reason
able to assume that, when this energy deficit
reaches the point where cellular integrity can no
longer be maintained, cellular disorganization oc
curs, followed rapidly by the death of the cell.
This may explain the carcinocidal effect of 6-AN
on the 755 tumor.
The lack of a similar nucleotide shift in lung
tissue (Chart 1), as a result of 6-AN administra
tion, is not understood. A few relevant points,
however, should be discussed. 6-AN, although a
very powerful antagonist of vital DPN-dependent
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1958 American Association for Cancer Research.
DIETRICHet al.—6-Aminonicotinamîde
and Enzyme Systems
systems, in no case completely blocks an enzy
matic step. While the animal is alive, a residual
amount of activity persists. Thus, it can be as
sumed that the resynthesis of ATP continues, but
at a greatly decreased rate. Certain mature tissues
have a lower energy requirement (ATP) than do
rapidly dividing tissues, such as the 755 tumor.
Consequently, two tissues could possess the same
ATP synthetic ability and be inhibited by 6-AN to
the same extent. However, if the energy require
ment (ATP) of the one tissue far exceeds the en
ergy requirement of the second tissue, gross bio
logical changes may occur in the tissue with the
greatest energy requirement, no changes being ob
served in the tissue with the lower energy require
ment. This may explain why lung, having the
same ATP synthetic capacities as the 755 tumor,
is not affected by 6-AN therapy.
These data indicate that, at least as far as these
model systems are concerned, quantitative bio
chemical differences are an important, if not the
most important, consideration in explaining the
selective action of 6-AN in destroying or slowing
the growth of the 755 tumor without producing
irreversible changes in other tissues of the host.
This enzymatic differential permits the inhibition
of the enzymatic activity by 6-AN in tissues hav
ing low DPN concentrations and low activities on
critical DPN-dependent enzymes without inhibi
tion occurring in tissues with higher co-factor con
centrations and enzymatic activities.
1279
Therapeutic doses of 6-AN (2 mg/kg for 8 days)
markedly inhibited the ability of tumor and lung
tissue to convert /3-hydroxybutyrate to acetoacetate and a-ketoglutarate to citrate. No other
tissues were affected by this level of 6-AN. Increas
ing the level of drug administered eventually in
hibited the /3-hydroxybutyrate and a-ketoglu
tarate systems in brain and liver tissue. At the
highest level of 6-AN, no effect was observed in
kidney and cardiac and skeletal muscle. In the tis
sues antagonized, tissues with lower DPN concen
tration and lower DPN-dependent enzymatic ac
tivities were inhibited by low levels of 6-AN. Tis
sues such as liver which are protected by higher
enzymatic and DPN levels required the adminis
tration of higher levels of 6-AN before enzymatic
inhibition is observed.
The concentration of 6-amino-DPN in the 755
tumor and six normal mouse tissues was deter
mined after the administration of a single injection
of 6-AN. Liver, kidney, and the 755 tumor syn
thesized 6-amino-DPN to the same degree. The
other tissues were found to have concentrations of
6-amino-DPN in the following order of decreasing
activity: brain, lung, heart, and skeletal muscle.
The administration of 6-AN caused a marked
lowering of the ATP and ADP levels and a large
increase in the concentration of AMP in the 755
tumor. Lung tissue under the same conditions ex
hibited no change in the acid-soluble nucleotide
pattern.
ACKNOWLEDGMENTS
SUMMARY
We
are
indebted
to Dr. Willard Johnson of Frank Horner,
The 755 tumor and six normal tissues of the
Ltd., Montreal, Canada, for generous supplies of 6-AN.
C57 mouse were analyzed for total DPN concen
tration and enzymatic capacities to oxidize ßREFERENCES
hydroxybutyrate,
a-ketoglutarate, and malate.
1. ACKERMANN,
W. W., and POTTER,V. R. Enzyme Inhibi
The DPN levels of these tissues had the following
tion in Relation to Chemotherapy. Proc. Soc. Exper. Biol.
&Med.,72:l-9,
1949.
order of decreasing concentration: liver, kidney,
2. AYENGAR,
P., and ROBERTS,E. Inhibition of Utilization of
heart, brain, skeletal muscle, lung, and tumor.
Glutamic Acid by Lactobacillus arabinosus. Proc. Soc. Ex
These values fell into two separate magnitudes
per. Biol. & Med., 79:476-81, 1952.
with heart, liver, and kidney having roughly 3 3. BALLANTYNE,
R. M., and McIÕENRY,E. W. Vitamin Bs
times the concentration of total DPN as did skele
and Biotin in Human Cancer Tissue. Cancer Research,
9:689-91, 1949.
tal muscle, lung, brain, and the 755 tumor. The
4.
BOYLAND,E. Chemical Carcinogenesis and Experimental
ability to convert jS-hydroxybutyrate to acetoChemotherapy of Cancer. Yale J. Biol. & Med., 20:821^tl,
acetate, and a-ketoglutarate and malate to citrate,
1948.
was found to be parallel in all tissues studied, the
5. DIETRICH,L. S.; KAPLAN,L. A.; and FRIEDLAND,I. M.
Pyridine Nucleotide Metabolism: Mechanism of Action
highest activity being found in the liver. The other
of the Niacin Antagonist, 6-Aminonicotinamide. J. Biol.
tissues showed the following order of decreasing
Chem., 233:964-68, 1958.
activity: kidney, brain, heart, tumor, lung, and
6. DIETRICH,L. S., and SHAPIRO,D. M. Antagonist Studies:
skeletal muscle.
Comparison of Deoxypyridoxine and Pyridoxine Deficient
The /3-hydroxybutyrate system was more sensi
Therapy on Various Vitamin B8 Enzymes. Proc. Soc. Ex
per. Biol. & Med., 84:555-58, 1953.
tive to 6-AN therapy than the system that con
7.
. Combination Chemotherapy of Cancer: Potentiaverts a-ketoglutarate to citrate. Malate conversion
tion of Carcinostatic Action of 8-Azaguanine by a Riboto citrate was found to be very resistant to antago
flavin Analog. Cancer Research, 13:699-702, 1953.
nism to 6-aminonicotinamide (6-AN).
8.
. In Vitro Effect of Two Pantetheine Analogues on
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1958 American Association for Cancer Research.
1280
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Cancer Research
Co-Enzyme A Requiring Systems. Proc. Soc. Exper. Biol.
&Med., 93:191, 1956.
FRIEDLAND,I. M.; KAPLAN,L. A.; DIETRICH,L. S.; and
SHAPIRO,D. M. 6-Aminonicotinamide Antagonism of
Pyridine Nucleotide Metabolism: Effect on the Acid
Soluble Nucleotides of Tumor Tissue. Fed. Proc., 17:224,
1958.
GREENSTEIN,J. P. Biochemistry of Cancer, pp. 175-315.
New York: Academic Press, Inc., 1947.
HIGGINS,H.; MILLER,J. A.; PRICE, J. M.; and STRONG,
F. M. Levels and Intracellular Distribution of Coenzyme
A and Pantothenic Acid in Rat Liver and Tumors. Proc.
Soc. Exper. Biol. & Med., 75:462-65, 1950.
JEDERKIN,L. A., and WEINHOUSE,S. Metabolism of Neoplastic Tissue. VI. Assay of Oxidized and Reduced DPN
in Normal and Neoplastic Tissue. J. Biol. Chem., 213:27180, 1955.
JOHNSON,W. J., and McCoLL, J. D. 6-Aminonicotinamide
—¿a
Potent Nicotinamide Antagonist. Science, 122:834,
1955.
LENTA,M. P., and l!11n i. M. A. The Coenzyme I Oxidase
System in Normal and Tumor Tissue. Cancer Research,
12:498-507, 1952.
MONTREVIL,J., and BOULANGER,P. Chromatographie
quantitative sur papier des ribonucleotides. Compt. Rend.,
231:247-49, 1950.
NATELSON,S.; PINCUS,J. B.; and LUGOVOT,J. K. MicroEstimation of Citric Acid; a New Colorimetrie Reaction
for Pentabromoacetone. J. Biol. Chem., 176:745-50, 1949.
OLSON,R. E. Oxidation of C'Mabeled Carbohydrate Inter
mediates in Tumor and Normal Tissue. Cancer Research,
11:571-84, 1951.
POTTER, V. R. Studies on the Reactions of the Krebs
Citric Acid Cycle in Tumor with Homogenates, Slices and
in Vim Techniques. Cancer Research, 11:565-70, 1951.
POTTER,V. R., and ELVEHJEM,C. A. A Modified Method
for the Study of Tissue Oxidations. J. Biol. Chem., 114:
495-504, 1936.
SCHNEIDER,W. C., and HOGEBOOM,G. H. Intracellular
Distribution of Enzymes. VI. The Distribution of Succinoxidase and Cytochrome Oxidase Activities in Normal
Mouse Liver and in Mouse Hepatoma. J. Nat. Cancer
Inst., 10:969-75, 1950.
SIEKEVITZ,P., and POTTER,V. R. Biochemical Structure
of Mitochondria. II. Radioactive Labeling of Intramitochondrial Nucleotides during Oxidative Phosphorylation.
J. Biol. Chem., 216:237-55, 1955.
SHAPIRO,D. M.; DIETRICH,L. S.; and SHILS,M. E. Quan
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
Vol. 18, December, 1958
titative Biochemical Differences between Tumor and Host
as a Basis for Cancer Chemotherapy. II. Riboflavin. Can
cer Research, 16:575-80, 1956.
•¿ . Quantitative Biochemical Differences between Tu
mor and Host as a Basis for Cancer Chemotherapy. V.
Niacin and 6-Aminonicotinamide. Ibid., 17:600-604, 1957.
SHAPIRO,D. M., and FDGMANN,
R. A. Combination Chem
otherapy of Cancer with 8-Azaguanine and Riboflavin
Analog. Proc. Soc. Exper. Biol. & Med., 81:239-12, 1952.
SHAPIRO,D. M.; FUGMANN,R. A.; and DIETRICH,L. S.
Partial Regression of a "Solid" Tumor with Multi-Com
pound Chemotherapy. Cancer Research, 17:1067, 1957.
SHAPIRO,D. M. ; KREAM,J. ; and DIETRICH,L. S. Combina
tion Chemotherapy of Cancer: Potentiation of Carcinostatic Activity of 8-Azaguanine by 6-Formylpteridine.
Proc. Soc. Exper. Biol. & Med., 81:616-19, 1952.
SHAPIRO,D. M.; SHILS,M. E.; and DIETRICH,L. S. Quan
titative Biochemical Differences between Tumor and Host
as a Basis for Cancer Chemotherapy. I. Vitamin Be. Cancer
Research, 13:703-8, 1953.
SHAPIRO,D. M.; SHILS, M. E.; FUOMANN,R. A.; and
FRIEDLAND,I. M. Quantitative Biochemical Differences
between Tumor and Host as a Basis for Cancer Chemo
therapy. IV. Niacin and 2-Ethylamino-l,3,4-Thiadiazole.
Cancer Research, 17:29-33, 1957.
SHILS,M. E.; FRIEDLAND,
I. M.; FINE, A. S.; and SHAPIRO,
D. M. Quantitative Biochemical Differences between Tu
mor and Host as a Basis for Cancer Chemotherapy. III.
Thiamine and Coenzyme A. Cancer Research, 16:581-84,
1956.
STERNBERG,
S. S., and PHILIPS,F. S. 6-Aminonicotinamide
and Acute Degenerative Changes in the Central Nervous
System. Science, 127:644-15, 1958.
TSUBOI,K. K., and PRICE,T. D. Isolation, Detection and
Measure of Microgram Quantities of Labeled Tissue Nu
cleotides. Arch. Biochem. & Biophys. (in press).
WALKER,P. G. A Colorimetrie Method for the Estimation
of Acetoacetate. Biochem. J., 68:699-704, 1954.
WEINHOUSE,S. ; MILLINGTON,
R. H. ; and WENNER,C. E.
Metabolbm of Neoplastic Tissue. I. The Oxidation of Car
bohydrates and Fatty Acids in Transplantable Tumors.
Cancer Research, 11:845-50, 1951.
WENNER, C. E.; SPIRTES,M. A.; and WEINHOUSE,S.
Enzymes of the Citric Acid Cycle in Tumors. J. Am. Chem.
Soc., 72:4333, 1950.
WILLIAMS,R. J. B Vitamins and Cancer, pp. 253-66.
A.A.A.S. Research Conference on Cancer. Lancaster, Pa.:
Science Press Printing Co., 1945.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1958 American Association for Cancer Research.
Quantitative Biochemical Differences between Tumor and Host
Tissue: VI. 6-Aminonicotinamide Antagonism of DPN-dependent
Enzymatic Systems
L. S. Dietrich, Leatrice A. Kaplan, Ira M. Friedland, et al.
Cancer Res 1958;18:1272-1280.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/18/11/1272
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1958 American Association for Cancer Research.