6AN, (
)an analogue of niacin, is currently being
evaluated in preclinical trials as part of a multidrug anti-cancer
regimen(1, 2) , having undergone clinical trials as a
single agent in the 1950s(3) . It has been shown to have
activity as a radiosensitizer in vitro(4) and be a
potentiator of 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity both in vitro and in vivo(5) . 6AN can compete
with niacin in the synthesis of NAD(P), being metabolized to 6ANAD or
6ANADP, which, in turn, can act as competitive inhibitors of
NAD(P)-requiring processes, such as generation of ATP by oxidative
phosphorylation. 6ANADP is a particularly potent inhibitor of the PPP
enzyme 6PG dehydrogenase. This leads to cytotoxicity by inhibition of
nucleotide synthesis(6, 7) , poly(ADP-ribose)
synthesis (5) and of glycolysis(8) .
Fig. 1summarizes the major pathways of glucose metabolism in
tumor cells. Tumor cells have long been known to metabolize glucose to
lactate, via glycolysis, under aerobic conditions (9) . A
significant portion of glucose, however, has been shown to be
metabolized to lactate via PPP(10, 11) . The PPP
provides NADPH for biosynthetic processes and ribose sugars for
nucleotide synthesis. Glucose 6-phosphate can be metabolized to
fructose 6-phosphate via phosphoglucose isomerase as part of glycolysis
or enter PPP, proceeding via the enzyme glucose 6-phosphate
dehydrogenase.
Figure 1:
Schematic of glucose
metabolism via glycolysis and the pentose phosphate pathway
highlighting the metabolites and enzymes of importance in this study.
The site of inhibition by 6AN, as 6ANADP, is marked by the double
diagonal. 1, hexokinase; 2, G6P dehydrogenase; 3, 6-phosphoglucono-
-lactonase; 4, 6PG
dehydrogenase; 5, phosphopentose
isomerase.
C-NMR has been widely used to
investigate the pathways of cellular glucose metabolism, both in
vitro and in vivo as well as in
humans(11, 12, 13, 14, 15, 16, 17, 18, 19) .
The effect of a number of drugs and hormones on glucose and glycogen
metabolism have been studied, such as vasoactive intestinal
peptide(20) , rhodamine 123 (21) and
lonidamine(22) . Most of these studies have been limited to
observation of glucose utilization and glycogen and lactate production,
although labeled alanine and glutamate have also been observed in some
systems. In this study we have investigated the effect of 6AN on the
glucose metabolism of RIF-1 tumor cells using
C and
P NMR, and observed accumulation of the PPP intermediates
6PG and 6PGL. We also investigated the potential of 6AN as a
radiosensitizer.
MATERIALS AND METHODS
Cells
RIF-1 cells were grown in RPMI 1640
supplemented with 10% fetal calf serum (Intergen, Purchase, NY). 96 h
before the start of perfusion, approximately 5
10
RIF-1 cells were seeded on 0.12 g of conditioned Cultispher-G
collagen beads (Hyclone Laboratories, Inc., Logan, UT) in a spinner jar
in 125 ml of medium. The medium for perfusion experiments was
phosphate-free RPMI 1640 supplemented with 10% fetal calf serum and 1.2
mg/liter DNase I (Sigma). The total number of cells on the beads at the
time of transfer from the spinner flask to the perfusion apparatus was
approximately 1.7
10
cells. Cells were counted by
removing 1 ml of beads and medium from the spinner flask. Following
sedimentation of the beads 0.8 ml of the supernatant was removed and
replaced by 0.8 ml of collagenase (10 mg/ml). This mixture was
incubated at 37 °C for 30 min. With the collagen beads digested, it
was possible for the released cells to be counted by eryrthrosin B dye
exclusion. The total volume of the perfusion circuit, including the
reservoir, was 130 ml. The head space of the medium reservoir was
gassed with O
/CO
(95:5). The design of the
perfusion apparatus was as described previously for agarose
threads(23) . Cells were perfused on the collagen beads as a
fluidized bed in a shortened screw cap NMR tube (Wilmad, Buena, NJ).
The reduction in upward force as the cross-sectional area of the tube
increased at the neck was sufficient to prevent flow of cells into this
part of the tube; the medium flow rate was adjusted accordingly.
NMR
Two five-turn solenoid coils, wrapped around
the bead-containing volume, were used to collect either
P
NMR spectra or
C NMR spectra. Proton-decoupled
P NMR spectra were obtained using a WALTZ-16
pulse-sequence on a Bruker/GE 4.7T Omega spectrometer (Bruker NMR,
Fremont, CA) operating at 81.03 MHz.
P NMR data were
acquired with a spectral width of 10,000 Hz, a 60° pulse angle
(12-µs pulse width), a relaxation delay of 2 s, 4K data points, and
4096 signal-averaged free induction decays. Under these conditions
spectra were partially saturated. 135 min of data acquisition (4096
transients) was sufficient to give a good signal:noise ratio.
P NMR spectra were processed with 5-Hz exponential line
broadening and referenced to PCr at 0 ppm. Proton-decoupled
C NMR spectra of cells were acquired at 50.34 MHz.
C NMR spectra of cells were acquired with a sweep width of
20,000 Hz, a 60° flip angle (30 µs), a recycle time of 3 s,
8192 data points, and 1024 transients. Each spectrum took 55 min to
acquire.
C NMR spectra were processed with 2-Hz
exponential line broadening and referenced to the
-anomer of
glucose at 96.0 ppm, except where stated in the figure legends. As a
known amount of labeled glucose was used in each experiment, it was
used as an internal concentration standard. Since two different coils
were used it was necessary to retune and shim the magnet each time the
observe nucleus was changed. The NMR tube containing collagen beads was
immersed in water to minimize inhomogeneities due to magnetic
susceptibility gradients(24) . A Helmholtz decoupling coil was
built on the outside of the water bath.High resolution
proton-decoupled
C and
P NMR spectra of cell
extracts, at approximately pH 9, were acquired on a Bruker AC 250-MHz
magnet operating at 62.90 and 101.25 MHz, respectively, using a 5-mm
probe. Experimental parameters for
C NMR were 20,000 Hz,
64K data points, a 60° flip angle (pulse width of 3.0 µs), and
a recycle time of 4.1 s. Spectra were zero-filled and processed with
1-Hz line broadening. Spectra were referenced to the
-anomer of
glucose at 96.0 ppm. Experimental parameters for
P NMR
included a sweep width of 15,000 Hz, 32K data points zero-filled to
64K, a 45° flip angle of 2.75-µs pulse width, and a recycle
time of 5.1 s. Free induction decays were processed with 1-Hz
exponential line broadening. Spectra were referenced to GPC at 0.0 ppm,
as the chemical shift of this peak is almost invariant with changes in
pH(25) . Dimethyl methyl phosphonate (1 mM) was used
as an internal concentration standard for calculating cellular
concentrations of phosphatic metabolites. It was assumed for these
calculations that the density of the extracted cells was the same as
that of the deuterium oxide in which the extract was reconstituted.
Assignments of peaks in
P NMR spectra of PCA extracts
were made by adding known compounds to the extract and noting whether
they were coincident with peaks present in the extract spectrum. In
some cases assignments were confirmed by pH titration.
Experimental Protocol
After the collagen beads had
been transferred to the perfusion apparatus, the magnet shimmed and the
coil tuned and matched, two
P NMR spectra were collected.
Following this initial stabilization period, the medium reservoir was
replaced by 100 ml of phosphate-free perfusion medium with
[1-
C]glucose (180 mg) substituted for
[U-
C]glucose. This volume of labeled media was
much greater than the volume of media already in the perfusion circuit,
so labeled and unlabeled pools were able to equilibrate during shimming
and tuning. The total volume of the medium reservoir and the perfusion
circuit was 130 ml. Sixteen sequential
C NMR spectra were
then acquired over the next 15 h.
C label was then washed
out of the perfusion apparatus by perfusing with two aliquots of
unlabeled media, each for 15 min. A
P NMR spectrum was
collected before once more changing over to media containing
[1-
C]glucose (180 mg) and a final concentration
of 40 µM 6AN. Sixteen
C NMR spectra were
acquired as before. Following wash-out of the label and 6AN, further
C NMR spectra were acquired. It was noted that the peaks
arising due to the C-1 carbons of glucose were reduced to natural
abundance levels, indicating efficient removal of label and drug.
Finally a
P NMR spectrum was acquired prior to harvesting
the cells for PCA extraction.
C and
P NMR
data were collected from a total of three separate perfusion
experiments in this manner.
P NMR spectra were obtained,
in these experiments, immediately before and after each period of
perfusion in the presence of [1-
C]glucose, with
C NMR data acquired at all other times.In addition to
these experiments, three further experiments were carried out in which
RIF-1 cells were perfused, both with and without 6AN, in the absence of
labeled glucose. The same protocol, as described above, was followed
for experiments in which
P NMR spectra were acquired
throughout. Unlabeled glucose was used in these experiments. Initially
eight
P NMR spectra were acquired prior to addition of
6AN, after which a further six spectra were acquired. Following
wash-out of the drug, six more
P NMR spectra were
acquired. In three control experiments, in which drug was not added
after 22 h, fresh perfusate was exchanged for the old at this point,
and perfusion continued for an additional 22 h, for a total of 44 h.
Perchloric Acid Extracts
Chelex-100 was obtained
from Sigma. All other reagents were of analytical grade. PCA extracts
of excised tumors were made using slight modifications of previously
described methods(26) . PCA extracts of cells on beads were
made by washing sedimented beads directly into liquid nitrogen with a
small volume of medium. Briefly, the cells and beads were ground to a
powder in a liquid nitrogen-cooled mortar. Approximately 3 volumes of
10% PCA was added to the powdered cells. This mixture was ground
further until cells and PCA were well mixed. The mixture was then
transferred to a polypropylene centrifuge tube and thawed and
immediately refrozen three times. Following this freezing and thawing
cycle the precipitate was removed by centrifugation for 5 min. The
supernatant was removed and neutralized with potassium carbonate (1 N). Following adjustment to pH 10.0, the potassium perchlorate
precipitate was removed by centrifugation. The supernatant was then
mixed and regularly shaken, over a period of 1 h, with Chelex-100 to
remove any paramagnetic ions. Chelex-100 was subsequently removed by
centrifugation. The supernatant was frozen and lyophilized prior to NMR
analysis. The lyophilized extract was reconstituted in 1-2 ml of
deuterated water.
Medium Extracts
A crude extraction procedure,
similar to that described previously(27) , was carried out to
concentrate the media and remove serum protein. Briefly, an equal
volume of cold acetone was added to the harvested media sample and left
on ice for 20-30 min. The precipitated protein was removed by
centrifugation at 1500
g for 10 min. The supernatant
was decanted from the protein fraction, and the acetone was removed
from the sample under vacuum using a rotary evaporator. The temperature
of the water bath used was not in excess of 37 °C. Chelex-100
(Sigma) was added to the remaining water phase and shaken periodically
over a period of approximately 2 h to chelate any paramagnetic ions
present in the sample. Chelex-100 was removed by centrifugation, and
the resulting solution was lyophilized. The lyophilized powder was
reconstituted in 2 ml of deuterated water.
Surviving Fraction Experiments
10
RIF-1 cells/ml were seeded in 5 ml of RPMI 1640, supplemented
with 10% fetal calf serum in 25-cm
culture flasks and grown
for 48 h, in order to establish log phase growth. After 48 h the media
was changed. Different flasks of cells were harvested by trypsinization
following one of eight different treatments. Control cells were
harvested without any treatment. Three flasks were treated with 40
µM 6AN for 15 h as in the NMR experiments. Of these, one
flask was harvested after 15 h, one was exposed to 3-Gy irradiation
after 15 h of 6AN exposure, and the other was exposed to 6-Gy
irradiation. Irradiation was carried out as described previously. These
irradiated flasks of cells were harvested 3 h following
irradiation(23) . These conditions were reversed in other
experiments, with the two doses of irradiation preceding the 6AN
treatment by 3 h. Also cells were exposed to 3- or 6-Gy irradiation
only and harvested 3 h later. The trypsinized cells were counted and
plated on a 5-cm Petri dish in 5 ml of RPMI 1640 supplemented with 10%
fetal calf serum, 2 mM glutamine, and nonessential amino
acids. A number of plating densities were used to ensure colony counts
were in a range likely to give accurate data. After 9 days the plates
were stained for 3 h with crystal violet and rinsed, and surviving
colonies were counted. All results are quoted as mean ± 1
standard deviation of triplicate determinations.
Statistics
Paired t tests were used to
evaluate differences in the metabolic response of batches of cells to
perfusion in the presence and absence of 6AN. Unpaired t tests
were used to compare differences between cells that were exposed to 6AN
after 22 h and those that were perfused for 48 h in the absence of 6AN
and to compare plating efficiencies in the surviving fraction
experiments. Errors are quoted as plus or minus one standard deviation. Error bars on the figures are standard errors of the mean.
RESULTS
P NMR Experiments
Fig. 2shows
P NMR spectra of RIF-1 cells
obtained at intervals during a perfusion experiment, following the
addition of 6AN. These spectra demonstrate a decrease in PCr and an
accumulation of 6PG following the addition of 6AN (40 µM)
to the perfusate. It can be seen from Fig. 3that this
accumulation of 6PG continued for at least 12 h after wash-out of 6AN,
albeit at a reduced rate. This 6PG peak persisted for at least 36 h
following wash-out of 6AN. The extracellular concentration of inorganic
phosphate was constant in all of the experiments, as the same batch of
serum was used throughout. Also, the extracellular volume was much
larger than the intracellular volume, so changes in P
with
varying cell number were minimal. It was possible, therefore, to
normalize metabolite ratios for cell number by dividing the ratio by
the initial number of cells on the beads.
P NMR data were
obtained from cell perfusion experiments in which cells were perfused
both in the presence and absence of
C label (n = 7). There was a significant increase in PCr levels over
the first 22 h of perfusion in the absence of 6AN, the PCr/P
ratio increasing from 0.19 ± 0.04/10
cells to
0.36 ± 0.12/10
cells (p < 0.01). 20 h
after administration of 6AN, however, there was a significant drop in
this ratio to 0.12 ± 0.07/10
cells (p <
0.05). PCr/P
increased from 0.36 ± 0.10/10
cells to 0.42 ± 0.06/10
cells at 40 h, in
cells that continued to be perfused in the absence of 6AN (n = 3). PCr/P
was significantly lower after 40 h
of perfusion in cells exposed to 6AN than in control cells that were
not treated with the drug (p < 0.001). The observed
increases in NTP/P
were not significant. The initial
increase in PCr/P
, coupled with the disappearance of GPC
after 2-4 h, indicates that the cells were well perfused and not
stressed in the first 22 h prior to addition of the drug. The
maintenance of NTP and PCr levels for up to 48 h in untreated cells
confirm that it is valid to compare data from the same batch of cells.
Figure 2:
P NMR spectra of perfused
RIF-1 tumor cells on collagen beads. A, 1-5 h after the
addition of 40 µM 6AN; B, 6-10 h after the
addition of 6AN; C, 11-15 h after the addition of 6AN.
P NMR spectral peak assignments are as follows: 1, 6PG; 2, composite peak consisting of
phosphoethanolamine and NMP; 3, phosphocholine; 4,
inorganic phosphate; 5, phosphodiesters; 6, PCr; 7,
-NTP; 8,
-NTP; 9,
-NTP.
Figure 3:
A, plot showing the increase in
the 6PG:P
ratio after the addition of 40 µM 6AN to perfusate of perfused RIF-1 tumor cells on collagen beads.
The plot also follows the change in 6PG:P
after
wash-out of 6AN from the perfusate. B, plot showing change in
PCr/P
over time during perfusion of RIF-1 tumor cells in
which media were changed after 20 h to that containing regular media or
media containing 40 µM 6AN.
Identification of 6PG and 6PGL
After 4 h of perfusion of RIF-1 cells in the presence of
[1-
C] glucose and 6AN (40 µM) a
peak began to appear at around 178.8 ppm in the
C NMR
spectrum (see Fig. 4A). After a further 4 h, a second peak
appeared at 178.6 ppm. The downfield peak has been assigned to 6PG, by
observing an increase in the peak in both the
P and
C NMR spectra following addition of the authentic
compound. The peak assigned to 6PG in the
P NMR spectrum
was coresonant with added 6PG over a wide pH range, from pH 5.5 to 9.5.
The second peak, observed 0.2 ppm upfield from 6PG, was observed to
slowly disappear in extracts at neutral pH. The intensity of 6PG
increased as that of the second resonance decreased. 6PGL, the
precursor to 6PG in PPP, is known to spontaneously hydrolyze to 6PG in
solution at neutral pH even in the absence of
6-phosphoglucono-
-lactonase(28) . This peak was therefore
assigned to 6PGL, both on the basis of this evidence and the similarity
of its structure to 6PG. The rate of hydrolysis of 6PGL was much slower
at pH 9 than at neutral pH. 6PGL is not commercially available. Other
possible glucose metabolites were ruled out on the basis of their
chemical shifts. Further evidence for these assignments comes from the
fact that the chemical shifts of the two compounds reported here are
very similar to those reported for the nonphosphorylated
forms(29) . The chemical environment of the phosphate group in
these two molecules was sufficiently similar that they could not be
resolved in the
P NMR spectrum of extracts, even upon pH
titration of their chemical shifts.
Figure 4:
A, expansion of
C NMR
spectrum of RIF-1 tumor cells after 15 h of perfusion in the presence
of 10 mM [1-
C]glucose and 40 µM 6AN. B, expansion of the
C NMR spectrum of
RIF-1 tumor cells 2 h after wash-out of labeled glucose and 6AN. A and B were zero-filled and processed with 1-Hz
exponential line broadening. C, expansion of the
C NMR spectrum of PCA extract of RIF-1 tumors. The extract
was made 5 h after wash-out of
C label and 6AN. D, acetone extract of perfusate from RIF-1 tumor cells
perfused for 15 h in the presence of 10 mM [1-
C]glucose and 40 µM 6AN.
The chemical shift difference between the in vitro spectra and
the extract spectra is in part due to a pH difference but is also due
to the lack of intracellular interactions in
extracts.
C NMR Experiments
6PG and 6PGL
In each labeling experiment, RIF-1
cells were perfused for 15 h in the presence of
[1-
C] glucose. Following wash-out of this label
with unlabeled media, cells were perfused in the presence of both
[1-
C] glucose and 6AN. Each experiment,
therefore, acted as its own control. Fig. 4A shows a
C NMR spectrum acquired after 15 h of perfusion in the
presence of 1 mmol [1-
C]glucose and 40
µM 6AN, and Fig. 4B shows a
C
NMR spectrum following wash-out of
C-labeled media and
6AN, respectively. It can be seen that following wash-out of labeled
media, peaks arising due to 6PG and 6PGL persisted. Peaks due to
labeled glucose and lactate were reduced to natural abundance levels
(not shown). 6PG and 6PGL were diminished only slightly in the hours
following wash-out of the label and drug. This suggests that these
metabolites are intracellular. Fig. 4C shows a PCA
extract of RIF-1 cells after perfusion in the presence of
[1-
C]glucose and 6AN. Fig. 4D shows an acetone extract of the
C-labeled perfusate.
6PGL, but not 6PG, was found to be present. The presence of 6PG and
6PGL in PCA extracts of cells and the absence of 6PG in the perfusate
provide further evidence that these metabolites are intracellular. The
presence of 6PGL, but not 6PG, in the perfusate indicates that this
compound was more readily leaked from the cell. Cell lysis as a
mechanism for leakage of 6PGL can be ruled out, as this would be
expected to yield 6PG also. Neither 6PG nor 6PGL were observed in the
control experiments.The cellular concentration of 6PG calculated
from the
P NMR spectra of PCA extracts, made after the 6AN
wash-out phase of the perfusion experiment, was 2.0 µmol/10
RIF-1 cells. This was as compared with 0.3 µmol/10
RIF-1 cells of NTP. This value for the concentration of 6PG was
used to calculate the amount of unlabeled (1% natural abundance) 6PG
required to cause a doubling of the 6PG peak in the
C NMR
spectrum of a PCA extract of cells incubated with
[1-
C] glucose and 6AN. Addition of a 100
concentration of unlabeled 6PG caused an approximate doubling of the
peak assigned to 6PG in the
C NMR spectrum (since the
natural abundance of
C is approximately 1%), indicating
that it was essentially 100%
C-labeled. In the perfusion
apparatus the cells were restricted to a volume of 6 ml inside the NMR
tube. Assuming that 6PG and 6PGL are intracellular, these metabolites
are therefore restricted to this volume, whereas other labeled
metabolites such as glucose and lactate are present in the entire 130
ml of the perfusion apparatus. Fig. 5charts the increase in
concentration of 6PG and 6PGL following administration of 6AN, as
calculated from the
C NMR data. The corresponding increase
in 6PG/P
observed in the
P NMR spectrum over
the same period in an experiment in which
C label was not
utilized is shown in Fig. 3A. As expected, it can be
seen that 6PG appears at approximately the same time in both
experiments. The intracellular concentrations of 6PG and 6PGL after 15
h (900 min) of perfusion in the presence of 10 mM [1-
C]glucose and 40 µM 6AN
were 1.9 ± 0.8 µmol/10
RIF-1 cells and 0.8
± 0.4 µmol/10
RIF-1 cells, respectively. These
values are in close agreement with the value calculated from PCA
extracts.
Figure 5:
Plot showing accumulation of
C-labeled 6PG and 6PGL as measured from
C NMR
spectra of RIF-1 tumor cells perfused in the presence of 10 mM [1-
C] glucose and 40 µM 6AN.
Glucose and Lactate
Fig. 6shows two series
of
C NMR spectra. The first series in Fig. 6A shows the metabolism of glucose to lactate over a period of 15 h
in RIF-1 tumor cells perfused in the presence of 10 mM [1-
C]glucose. The second series in Fig. 6B shows glucose metabolism of the same cells over
15 h in the presence of 10 mM [1-
C]glucose and 40 µM 6AN. It
can be seen from these two series of spectra that metabolism of glucose
to lactate was reduced in the presence of 6AN. Fig. 7A shows
the change in glucose concentration over time in control and treated
cells. After 15 h the glucose concentration was 0.32 ± 0.03
mmol/10
cells and 0.46 ± 0.05 mmol/10
cells for control and treated cells, respectively. Glucose
utilization was therefore significantly inhibited in the presence of
6AN (p < 0.05). Similarly, Fig. 7B shows
that production of lactate was significantly inhibited (p < 0.05), being 0.17 ± 0.02 mmol/10
cells in control
and 0.07 ± 0.01 mmol/10
cells in the presence of
6AN.
H NMR analysis of the methyl peak of lactate and its
H-
C coupled satellites, in acetone extracts of
the perfusate, showed no significant change in the percentage of
labeling of lactate (data not shown). The amount of label unaccounted
for in the mass balance equation, i.e. lost as carbon dioxide,
was 1.67 ± 0.43 mmol/10
cells in control and 0.62
± 0.52 mmol/10
cells in the presence of 6AN.
Significantly less carbon was lost as carbon dioxide in cells treated
with 6AN than in control cells (p < 0.01).
Figure 6:
A,
series of 16
C NMR spectra of RIF-1 tumor cells perfused
in the presence of 10 mM [1-
C] glucose. B, series of 16
C NMR spectra of the same RIF-1
tumor cells as in A, perfused in the presence of 10 mM [1-
C] glucose and 40 µM 6AN.
C NMR spectral peak assignments are as follows: 1,
-anomer of glucose; 2,
-anomer of
glucose; 3, lactate.
Figure 7:
A, plot showing utilization of
[1-
C] glucose, as measured by
C
NMR, by RIF-1 tumor cells in the presence and absence of 40 µM 6AN. B, plot showing production of
[3-
C] lactate, as measured by
C
NMR, by RIF-1 tumor cells in the presence and absence of 40 µM 6AN.
Effect of 6AN on Radiosensitivity
The surviving fraction of RIF-1 cells was estimated following
exposure to 6AN and in combination with 3- or 6-Gy irradiation in order
to evaluate its potential as a radiosensitizer. The plating efficiency
of RIF-1 cells following the various treatments is summarized in Table 1. 6AN alone had no significant effect, but 3 and 6 Gy
caused significant cell kill with respect to control (both p < 0.05). The surviving fractions, relative to control, were 20
and 15% for 3 and 6 Gy, respectively. When 6AN was administered
following radiation there was no significant reduction in the plating
efficiency. When irradiation followed 6AN, however, there was a further
significant reduction in the surviving fraction. When 3- or 6-Gy
irradiation was followed by 6AN the plating efficiency was
significantly reduced compared with irradiation alone (p <
0.05 for 3 Gy, and p < 0.001 for 6 Gy), giving surviving
fractions relative to control of 11 and 6%, respectively. These data
indicate potentiation of radiotherapy by 6AN when this drug is
administered prior to x-irradiation.
DISCUSSION
Effect of 6AN on Pentose Phosphate Pathway
6AN
can compete with nicotinamide to form 6ANAD and 6ANADP, which are
competitive inhibitors of NAD(P)-requiring processes(30) . The
oxidative arm of PPP is an important source of ribose 5-phosphate,
which is required for nucleotide synthesis and of NADPH, which is
required as a reducing equivalent in many biosynthetic reactions.
6ANADP, therefore, can cause inhibition of glucose 6-phosphate
dehydrogenase and 6PG dehydrogenase, the NADP-requiring steps of this
pathway. The large accumulation of 6PG and the almost 3-fold decrease
in the amount of
C-label lost as carbon dioxide indicate a
strong inhibition of 6PG dehydrogenase. There was no detectable
accumulation of glucose 6-phosphate in vitro or in extracts,
in agreement with Keniry et al.(31) . As discussed
below, a significant increase in glucose 6-phosphate may have been
below the detection limits of this technique. While some inhibition of
glucose 6-phosphate dehydrogenase cannot be ruled out, the accumulation
of as much as 1.9 ± 0.8 µmol of 6PG/10
RIF-1
cells indicates that it was not significant relative to the inhibition
of 6PG dehydrogenase. The inhibitor constant of 6ANADP for 6PG
dehydrogenase has been shown to be at least 1 or 2 orders of magnitude
lower than that of glucose 6-phosphate dehydrogenase and other
enzymes(32, 33, 34) . It is possible,
therefore, that while 6PG dehydrogenase is strongly inhibited,
glucose-6-phosphate dehydrogenase could be relatively unaffected. Immersion of the NMR tube, containing the cells, and the NMR
detection coil in a water bath in order to minimize field
inhomogeneities due to magnetic susceptibility has enabled us to obtain
resolution in the
C NMR spectrum close to that of PCA
extracts (see Fig. 4) (24) . This is the first report in
which any accumulation of 6PGL has been noted. The large chemical shift
range of
C NMR has allowed us to readily resolve this
metabolite from the closely related metabolite 6PG in vitro.
It is possible that the structural similarity of 6PG and 6PGL precludes
the distinguishing of the two using other techniques, including
P NMR. 6PGL is the direct metabolic precursor of 6PG,
being hydrolyzed to 6PG via the enzyme 6PGL lactonase. Brodie and
Lipmann (28) noted that 6PGL was not formed when 6PG was
incubated in the presence of isolated yeast
6-phosphoglucono-
-lactonase, indicating that the equilibrium of
the enzyme lies overwhelmingly in favor of 6PG formation. This
suggests, therefore, that formation of 6PGL is probably due to
competitive feedback inhibition by 6PG. Further evidence for this comes
from the fact that the K
of the enzyme for 6PGL
has been found to be low, and the cellular concentration of 6PGL in
various tissues was also low (10 nmol/g of tissue)(35) . 6PG
and 6PGL continued to increase throughout the incubation period,
indicating that any inhibition of glucose 6-phosphate dehydrogenase,
whether by feedback or by 6ANADP, was necessarily limited as discussed
above. It has been shown in this study that 6PG and 6PGL are
intracellular, with only a small amount of leakage to the perfusate.
Like many other phosphorylated intermediates, 6PG and 6PGL transport
out of the cell is restricted, leading to a large accumulation. Only
the cyclic 6PGL was observed in acetone extracts of the perfusate. No
6PG, which is the open chain form of the molecule, was observed. The
absence of 6PG in the perfusate indicates that 6PGL was not leaked into
the perfusate via cell lysis. The similarity of the structure of 6PGL
to glucose (both are pyranoses in solution) suggests that it may have
reached the perfusate via the glucose transporter.
Effect of 6AN on Glycolysis
The observed reduction
in glucose utilization is not in itself an indication of inhibition of
glycolysis, as there was a significant flux of glucose carbon into PPP,
as demonstrated by the accumulation of 6PG and 6PGL. Production of
labeled lactate, however, is a direct measure of the glycolytic rate.
Lactate produced via PPP is not labeled, as the label at the C-1
position is lost as carbon dioxide; hence only lactate produced via
glycolysis is labeled at C-1. As can be seen from Fig. 7,
lactate production, as well as glucose utilization, was reduced in
cells exposed to 6AN after 200 min, and almost total inhibition
occurred after 400 min. The onset of this inhibition of glycolysis
correlated with the start of accumulation of 6PG. After 400 min, when
inhibition of glycolysis was effectively complete, the concentration of
6PG was 1.0 ± 0.6 µmol/10
RIF-1. 6PG has been
shown to cause inhibition of glycolysis, acting as a competitive
inhibitor of phosphoglucose isomerase(35) , and Kahana et
al. reported that the K
of 6PG for
phosphoglucose isomerase was 5 µM(36) . Although
some inhibition of phosphofructokinase by 6PG has been observed (37) the inhibition of phosphoglucose isomerase is more than
sufficient to explain the almost complete shutdown of glycolysis
following accumulation of 6PG. It is possible that 6PGL is in fact a
much stronger inhibitor of phosphoglucose isomerase, as its structure,
in the pyranose ring form, is much more similar to that of glucose
6-phosphate than is that of the open chained 6PG.This inhibition of
both glycolysis and PPP by administration of 6AN to RIF-1 tumor cells
caused a significant reduction in PCr/P
. The onset of PCr
depletion correlated with the first appearance of 6PG and the
associated inhibition of glycolysis. Inhibition of these pathways of
glucose metabolism prevents the cells from synthesizing NTP via
glycolysis and the Krebs cycle and NADPH via PPP. Inhibition of
glycolysis is especially important to tumor cells for which this
pathway is a major energy source(38, 39) . A lack of
NTP synthesis will lead to a depletion of NTP as it is catabolized for
maintenance energy (40) . This result is analogous to a study
in which ascites tumor cells were incubated with the glycolytic
inhibitor 2-deoxyglucose (41) and a previous study in which a
significant depletion of ATP was observed after 4 days of exposure to
6AN(42) . Although NTP/P
was not significantly
altered 24 h after administration of 6AN, PCr/P
was reduced
approximately 4-fold over the same period. Keniry et al. did
not observe any change in NTP/P
or PCr/P
after
24 h in the presence of 100 µM 6AN(31) . The cells
used in their experiments, however, were analyzed by NMR following
trypsinization rather than by using a perfusion apparatus. It has been
shown in human muscle that cellular PCr concentrations act as a buffer
for NTP in order to maintain the cellular NTP
concentration(43) . PCr, as a buffer for NTP levels, would be
expected to be severely depleted before any significant reduction in
NTP was observed, during inhibition of glycolysis. A number of
different mechanisms for the cytotoxicity of 6AN have been
demonstrated(5, 6, 7, 8) , and all
of these mechanisms could contribute to the effects observed here. The
inhibition of glycolysis, however, is sufficient to explain the
reduction of PCr/P
observed here.
6AN as a Radiosensitizer
The surviving fraction
experiments indicated that 6AN enhanced the effect of x-irradiation
when cells were exposed to 6AN for 15 h prior to irradiation. There was
no such effect when 6AN treatment followed x-irradiation, which may be
attributed to the fact that DNA repair was complete at 3 h before
addition of the drug, or when 6AN was given alone. These data indicate,
therefore, that 6AN alone has no significant cytotoxic effect but does
act as a radiosensitizer. 6AN inhibits 6PG dehydrogenase, which is
involved in the production of both ribose units and NADPH, both of
which are required for DNA repair and synthesis. 6AN, therefore, could
act as a radiosensitizer through the indirect inhibition of DNA repair,
which is required following x-irradiation. Alternatively, the reduction
in cellular PCr in the presence of 6AN, as measured by NMR, and the
increased efficacy of radiation treatment, suggest that 6AN may
potentiate other treatments, including radiation (4) and
1,3-bis(2-chloroethyl)-1-nitrosourea(5) , by reducing the
amount of cellular energy available for DNA repair.
Conclusions
This study demonstrates the value of
NMR for study of the effects of antimetabolites on cellular metabolism.
The use of
C NMR has enabled us to simultaneously monitor
the accumulation of 6PG due to the inhibition of PPP by 6ANADP and the
secondary inhibition of glycolysis. We were also able to identify 6PGL,
a metabolite that had not previously been observed in studies of the
drug 6AN using other techniques. 6AN has also been shown to be a
radiosensitizer, acting either by inhibition of ribose and NADPH
synthesis or by compromising cellular energy levels.