Download The Effects of Antibiotics and Cancer

[CANCER RESEARCH 33, 1670-1676, July 1973]
The Effects of Antibiotics and Cancer Chemotherapeutic Agents on
the Cellular Transport and Antitumor Activity of Methotrexate in
L1210 Murine Leukemia
Robert F. Zager, Shayla A. Frisby, and Vincent T. Oliverio '
Laboratory of Chemical Pharmacology, National Cancer Institute, N1H, Bethesda, Maryland 20014
SUMMARY
INTRODUCTION
MTX2 is widely used for the treatment of malignant
tumors in man (1, 6, 10, 16, 17). However, with the
exception of choriocarcinoma, relatively low response rates
of most malignant neoplasms to MTX as a single agent in
chemotherapy continues to be somewhat disappointing.
Prompted by the successful results of combination chemo
therapy in acute leukemia and the lymphomas (4, 9, 12),
MTX is now being used in combination with many other
cancer chemotherapeutic agents for the treatment of other
human malignant neoplasms. Patients being treated with
MTX often receive many other pharmacologically active
compounds concomitantly. Little work has been done to
determine what effect, if any, these other agents have on the
transport of MTX into tumor cells and ultimately on its
antitumor effect in vivo.
To examine this area, a series of experiments was
performed to evaluate the effects of certain commonly
administered "drugs" on MTX transport in the L1210
leukemia system. These studies have shown that certain
increased MTX transport by 66 and 49%, respectively, with chemotherapeutic agents and antibiotics can affect MTX
the effect of vincristine still being significant at 10~9 M. transport and possibly MTX antitumor effect in this system.
Efflux of MTX-3H from preloaded L1210 leukemia cells
was not significantly altered suggesting little effect by any of
these agents on intracellular binding of MTX to dihydrofo- MATERIALS AND METHODS
late reducÃ-aseenzyme.
The methodology used in these experiments has been
The transport inhibitors, hydrocortisone and L-asparagidescribed
in detail previously by Goldman et al. (8). It is
nase, antagonized the antitumor activity of MTX in L1210
leukemia-bearing mice. When hydrocortisone (175 mg/kg reviewed only in outline form except where modifications
i.p. every 4 days, five times) preceded MTX (25 mg/kg i.p. have been made or where new methods have been used.
Cells, Media, and IncubationTechniques. L1210 leukemia
every 4 days, five times) treatment by 180 min, the mean
cells
were grown in spinner culture containing 10% fetal calf
survival time was decreased by 7 days. Conversely, when
serum
(RPM1 1630 media). For experiments, cells in log
treatment with the transport enhancer vincristine (1.5
phase
growth
were prepared as previously described (8).
mg/kg i.p. every 4 days, four times) preceded MTX (25
Cells
were
suspended
in a buffer solution (124 mM NaCl, 16
mg/kg i.p. every 4 days, four times) by 30 min, the mean
m.MNaHCO3,
4
mM
KC1,
1.1 mM K2HPO4, 1.9 mM CaCl2,
survival time was increased from 23 to 43 days with 30%
and
1.0
mM
MgCl2)
at
pH
7.4 and placed in a glass
cures. The time course effects (interval between drug and incubation flask that was suspended
in a water bath at 37°
MTX doses) appear to implicate other factors in addition to
continuously agitated by a mechanical stirrer. A stream
transport effects in the antagonism or enhancement of and
of warmed humidified 95% O2-5% CO2 was passed over the
MTX antitumor activity in vivo. These studies suggest that
the concurrent use of cancer chemotherapeutic agents and incubation mixture.
The effect of the simultaneous addition of a number of
clinically useful antibiotics and cancer chemotherapeutic
agents on the transport of 2 x 10~6 M methotrexate
(4-amino-lO-methylpteroyl-glutamic acid; MTX) was stud
ied in L1210 murine leukemia cells in vitro. The results were
correlated with the effects of these agents on the antitumor
activity of MTX in BALB/cAnN x C57BL/KaLwN F,
mice bearing L1210 leukemia cells using optimal dose
levels and schedules.
Utilizing doses that are estimates of achievable peak
blood levels in man after conventional single i.v. doses,
corticosteroids, cephalothin, t.-asparaginase, and the Vinca
alkaloids showed the greatest effects on MTX uptake by
L1210 leukemia cells, while hydroxyurea, bleomycin, peni
cillin, and kanamycin appeared to have lesser but significant
activities. For example, hydrocortisone at 10~5and 10~3M
inhibited MTX transport 20 and 65%, respectively. Compet
itive and dose-related inhibition was clearly evident for this
agent. Vincristine and vinblastine at 10~5 M uniquely
antibiotics may significantly alter cellular transport and
ultimately antitumor activity of MTX.
1To whom requests for reprints should be sent, at this address.
Received October 5, 1972; accepted April 16, 1973.
1670
Measurement of MTX Uptakes. For experiments involv-
'The abbreviations used are: MTX, methotrexate (4-amino-10methylpteroyl-glutamic acid); DHFR, dihydrofolate reducÃ-ase;VCR,
vincristine; VBL, vinblastine.
CANCER
RESEARCH
VOL. 33
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M TX Interactions
ing a "drug," the "drug"-L1210-buffer
mixture was mixed
for 1 to 2 min in the incubation flask prior to the addition of
MTX-3H. Serial samples (2 to 3 ml each) were then
withdrawn at designated time intervals and were expelled
into 13-ml iced centrifuge tubes, and the cells were isolated
as a pellet by rapid centrifugation and disposal of the
supernatant material. The pellet was washed 2 times with
cold 0.9% NaCl solution and dried overnight at 80°.To
determine cellular MTX the pellets were weighed on a Cahn
G-2 electrobalance and digested with 0.4 ml of l N KOH at
80°for I hr, and a 0.2-ml aliquot was added to 18 ml of
scintillation solution (7) for counting in a Beckman LS-133
liquid scintillation spectrometer. Trypan blue exclusion tests
showed cell viability to be greater than 85% during in vitro
uptake experiments.
Efflux Experiments. Efflux experiments were performed
by preloading the L1210 with MTX-3H for 20 min then
washing 2 times with cold 0.9% NaCl solution, resuspending
in MTX-free buffer, and serially sampling as described
above. Other experiments evaluated the effect of "drugs" on
efflux by having these compounds present in the MTX-free
buffer during efflux.
Measurement of Unidirectional Influx. Unidirectional
flux measurements, which did not exceed the binding
capacity of DHFR in L1210 cells, were done as described by
Goldman et al. (8). Incubations were terminated by rapid
centrifugation followed by 2 ice-cold 0.9% NaCl solution
washes. Cell pellets were then processed as described above.
In Vivo Experiments. The L1210 leukemic ascites line
used in these studies was maintained in this laboratory in
BALB/cAnN x C57BL/KaLwN F, CDF, (1) mice by
weekly i.p. passage. CDFi female mice, 10 to 12 weeks old
and weighing 17 to 21 g/mouse, were used in these experi
ments. Drugs were dissolved in 0.9% NaCl solution and
injected i.p. in a volume of 0.01 ml/g body weight.
Experiments to determine the number of ascites cells per
mouse were done by sacrificing the animal on the appropri
ate day and repeatedly lavaging the peritoneal cavity with 3to 4-ml aliquots of 0.9% NaCl solution. After osmotic lysing
of the red blood cells, the number of cells was determined by
using a Coulter counter.
Chemicals. MTX-3',5'-3H was obtained from Amersham/Searle Corp., Des Plaines, 111., or Dhom, North
Hollywood, Calif., MTX-3H was purified by DEAE-cellulose column chromatography as previously reported (8).
Antibiotics were obtained from Calbiochem (Los Angeles,
Calif.) except for cephalothin and gentamicin for which
the clinical preparations were used. Chemotherapeutic
agents were obtained from the Drug Research and Devel
opment Branch of the National Cancer Institute.
RESULTS
Effect of "Drugs" on MTX-3?! Uptake by L1210 Leu
kemia Cells in Vitro. The "drugs" evaluated for their effect
on MTX transport in L1210 leukemia cells are listed in
Table 1. Drug concentrations are estimates of achievable
plasma levels in man following conventional single i.v.
with Other Drugs
doses. Corticosteroids, cephalothin, L-asparaginase, and the
Vinca alkaloids showed the greatest effects, and they are
discussed in more detail in subsequent sections. Other
"drugs" that appeared to have a significant effect on MTX
transport include hydroxyurea and bleomycin in the chemotherapeutic category and penicillin and kanamycin in the
antibiotic group. The Vinca alkaloids were particularly
interesting because of their unique ability to enhance MTX
transport.
Inhibitorsof MTX Transport in 1.1210 Leukemia Cells in
Vitro. Cephalothin inhibited the transport of MTX into
L1210 leukemia cells in vitro. Table 2 shows the doseresponse of this effect. At 10 3M(418 /ig/ml) inhibition was
46.5% of control uptake at 40 min of incubation. At 10~5M
(4.18 /ig/ml) little if any effect on MTX transport was seen.
Of the "drugs" evaluated, hydrocortisone was the most
effective inhibitor of MTX transport in the L1210 system.
At 10~5 M (3.6 ¿ig/ml)inhibition was 19.6% of control and
at 10~3 M (360 /ig/ml) the inhibition reached 62.8%. The
time course of this inhibition and its dose-response charac
teristics for hydrocortisone (sodium succinate) are shown in
Chart 1. The same results were seen for the free base of
hydrocortisone. As the concentration of hydrocortisone
increased from I0~eto 10"3 Mprogressively more inhibition
of MTX transport was seen. Saturation of the intracellular
DHFR binding sites with MTX (intracellular MTX level of
approximately 5 to 6 nmoles/g dry cell weight) was achieved
within 2 min in the control experiment (8). This time is
gradually prolonged by the inhibitory action of hydrocortisone so that at a concentration of IO"3 M saturation takes
approximately 40 min. Experiments evaluating the effect of
hydrocortisone on MTX transport during the period of
unidirectional influx, before DHFR is saturated, clearly
show that hydrocortisone can inhibit the unidirectional
transport of MTX.
Methylprednisolone was also an inhibitor of MTX trans
port but it was less effective than hydrocortisone at
equimolar concentrations. Dexamethasone was not an in
hibitor in this system.
As shown in Table 2, L-asparaginase is a relatively weak
inhibitor of MTX transport in L1210 leukemia cells. The
L1210 leukemia used in these experiments showed no
antitumor response to L-asparaginase therapy.
Enhancement of MTX Transport in 11210 Cells in Vitro.
The effects of the Vinca alkaloids on MTX transport in
L1210 cells were unique for the "drugs" examined in this
study. VCR and VBL markedly increased the uptake of
MTX in vitro to above control values. The dose-response of
this effect can be seen in Table 2. At equimolar concentra
tions VCR is a more effective enhancer of MTX transport
than is VBL. The VCR effect is still noted at 10~9 M
(0.00083 ¿ig/ml). The enhancing activity of the Vinca
alkaloids was observed at substantially lower concentrations
than the previously discussed inhibitory effects of cephalo
thin, hydrocortisone, and L-asparaginase. However, this
fact must be evaluated in light of the clinical fact that Vinca
alkaloids are routinely administered in significantly smaller
doses than are the above-mentioned inhibitors.
Further experiments have revealed that preincubation of
JULY 1973
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1671
R. F. Zager, S. A. Frisby, and K. T. Oliverio
process. It was seen that the inhibitors caused a fall in the
intracellular MTX level in proportion to their previously
discussed potency as inhibitors of MTX transport in vitro.
For example, addition of hydrocortisone, the most potent
concen
inhibitor of MTX transport examined, resulted in the efflux
tration
A in uptake
of all the "free" MTX in the L1210 cell, leaving behind only
(/ig/mi)»36250a752005037.52.5353035301040351.00.11502.5152523209.128.25%
vs.
control0-35
DrugHydrocortisonei.-AsparaginaseCephalothinHydroxyureaBleomycinMcthylprednisolonePenicillineKanamycin5-MercaptopurineNeomycinStreptomycinProcarbaz
that MTX that is presumably bound to DHFR. L-AsparagiTable I
Effect ofchemotherapeulic agents and antibiotics on MTX-3H uptake" in
LI210 leukemia cells in vitro
±0.4-30
1.4-25
±
0.5-16±-15
±
Table 2
Effect of "drugs" on MTX-3H uptake in LI210 leukemia cells in vitro"
±-I4±-12±-12±-8
inhibition
ofMTX-3H
uptake62.8
DrugHydrocortisone(sodium (moles/liter)1
±-7
±-4±-3±-3±-3
IO-35
x
1.654.8
±
IO-41
x
Buccinate)CephalothinMethylprednisolone(sodium
1.734.5
±
IO-41
x
2.319.6
±
IO'51
x
3.246.5
±
±-3
±(-1
mustardActinomycin
IO-35
x
1.630.2
±
±-1
DCytoxanChlortet
IO'41
x
2.014.8
±
±.1.7.1.9.5.5.0.3.5.2.0).8.2.2+
IO-41
x
1.86.2
±
racyclineGentamicinErythromycinCytosine
0.8+
1 ±
IO'51
x
2.545.2
±
1.5+
2 ±
0.9+4±
2 ±
IO-35
x
1.638.5
±
arabinosideDaunorubicinVBLVCRDrug
1.6+
x
succinate)L-AsparaginaseVBL IO-41
1.513.5
±
±0.8+49
5
IxO-42000"250"25»x
2.828.0
±
0.9+66
±
±0.9
l.l29.6±
1.87.1
±
"Uptakes were done with MTX-3H at 2.0 u»with an incubation of 40
±1.1%
min at pH 7.4 and 37°.
'Drug concentrations are estimates of achievable plasma levels in man
10-x
sulfateVCR
following conventional single i.v. doses.
10-x
•¿"Negative
values reflect inhibition of uptake. Positive values reflect
10-x
enhancement of uptake. All values mean ±S.E. Values represent the mean
10-x
of 3 to 4 separate experiments.
•¿"Measured
in units/ml.
10-x
sulfateConcentration
10-x
the L1210 cells with VCR or VBL (IO'5 M, pH 7.4, and
10-1
10-1
x
37°),for as short a time as 60 sec, followed by 0.9% NaCl
x 10-%
enhance
ment of
MTX-3Huptake93.7
3.449.0
±
1.117.6
±
1.714.8
±
2.065.6
±
1.733.0
±
4.029.3
±
3.936.6
±
1.22.4
±
±0.4
solution washes 2 times, will result in these cells having
enhanced transport of MTX in vitro. Incubating the cells
with VCR or VBL for as long as 20 min does not increase
the magnitude of this enhancing effect that approaches a
greater than 3-fold increase in the level of free intracellular
MTX compared to 40-min control values.
Effect of "Drugs" on MTX Efflux. Chart 2 shows the
efflux of MTX-3H from preloaded cells suspended in buffer
containing a "drug" but not MTX. None of the agents
caused significant alteration in the nonexchangeable level of
MTX, which suggests that none of these "drugs" altered
MTX binding to DHFR. Addition of "drug" to the buffer
did not significantly alter the form of the efflux curve. If
cells are first incubated with MTX and VCR and then
resuspended into MTX-free buffer in presence or absence of
VCR, the efflux rate and the nonexchangeable MTX levels
are comparable. Thus it would appear that VCR does not
alter the intracellular binding of MTX.
Addition of "Drug" to Bath duringUptake in Vitro. Chart
3 demonstrates the effect of inhibitors and enhancers on the
form of the control uptake curve when the "drugs" were
Chart 1. Inhibition of MTX-3H uptake by varying concentrations of
hydrocortisone (sodium succinate) in L1210 leukemia cells in vitro.
Extracellular concentration of MTX is 2 x 10"* Min all experiments. Each
added to the bath 20 min after initiation of the uptake
curve represents the average of 3 experiments.
1672
"All experiments performed with 2
incubation at 37°.
"Measured in units/ml.
IO'' M MTX and for 40 min of
Hydrocortisone 5x10 M
Hydrocortisone IO"3M
15
20
25
TIME (min)
CANCER
RESEARCH
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VOL. 33
MTX Interactions with Other Drugs
à Cepholothin
à Control
O——O
Vinblostme
levels of this agent. Also, transport inhibitors can cause loss
of cellular MTX from cells having intracellular levels
greater than that which is presumably bound to DHFR.
Kinetics of Transport Inhibition. When the transport
inhibitor hydrocortisone was present at constant external
concentrations and MTX was added over a range of
concentrations, the data can be plotted as the reciprocal of
the initial uptake velocity as a function of the reciprocal of
the concentration of MTX (see Chart 4). It was seen that the
line for the inhibitor showed increased slope when compared
to the control line and that the ordinate intercept was not
significantly changed, indicating competitive inhibition.
These experiments always measured unidirectional flux
since the level of intracellular MTX never exceeded the level
of DHFR in the cell.
Effect of "Drugs" on MTX Antitumor Activity in Vivo.
The effects of previously discussed inhibitors and enhancers
of MTX transport on the antitumor activity of MTX in the
L1210 murine leukemia were evaluated. Table 3 shows the
effect of pretreating the L1210 leukemia-bearing mice with
hydrocortisone i.p. prior to each dose of MTX. Hydrocorti-
( IO"4M)
»Hydrocorlisone
(IO"4M)
(IO"5M)
IO
rr
4
_L
J_
_L
J_
5
10
15
20
TIME
25
(minutes)
Chart 2. Effect of "drugs" on MTX efflux in LI2IO leukemia cells in
vitro. Variations of initial MTX level is noted because experiments were
done on different days with variations in the level of nonexchangeable
MTX. In all experiments, L1210 cells were preloaded for 20 min at 37°
with an extracellular concentration of MTX of 2 x IO"6 M. Each curve
16
represents the average of 3 experiments.
14 -
Vincristcne
28p
(IO'5M)
C 12
0>
Õ
9K
•¿o
o«10
o
/
C
Control
(MTX-2
«IO"6M1
Ë
(250
£ 8
in
L' Asporoginose
•¿
Cepholothin
(5x
units/ml)
IO"4M)
o /
/
e 6
<y
/
Hydrocorlisone
15«IO"<M)
A Extrocellulor
Hydrocortisone
B: Control
5x10
M
MTX
/
/o
10
20
30
TIME
40
60
(minutes)
Chart 3. Effect of "drugs" on MTX transport in L1210 leukemia cells
in vitro. Drugs were added to bath 20 min after initiation of the MTX
uptake process. Each curve represents the average of 3 experiments in
which the extracellular MTX was 2 x 10"* M.
nase, a relatively weak inhibitor of MTX transport, caused
only a slight decrease in the level of intracellular MTX.
VCR, an enhancer of MTX transport, caused a rapid rise in
the intracellular MTX to a level greater than twice control
values. From this experiment it was seen that VCR (and
VBL) has the potential to increase the level of free
intracellular MTX in cells that already have substantial
JULY
[MTXl
Chart 4. The effect of hydrocortisone on the unidirectional influx of
MTX in L1210 leukemia cells. Each point represents 3 experiments. At
each concentration of MTX only unidirectional flux was measured because
the maximum MTX concentration did not exceed the DHFR binding
capacity.
1973
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research.
1673
R. F. Zager, S. A. Frisby, and V. T. Oliverio
Table 3
Effect of hydrocorlisone (sodium succinole) and VCR sulfate on the survival of L12IO-bearing CDFl mice
treated with M TX
Mice were inoculated on Day 0 with 10'L12IO cells i.p. Hydrocortisone or VCR was always given prior to
MTX by stated interval. The difference ¡nmean survival between 60- and 180-min experiments (hydrocortisone) is significant (p < 0.01). Ten mice were used in each group.
MTX treat
mentControl25
mg/kgi.p..5
times25
5times25
mg/kg i.p.,
5times25
mg/kg i.p.,
between
treatmentHydrocortisone "drug"
MTX60
and
182,6,IO, 14,
(175mg/kg)
5xHydrocortisone
i.p.,
(175mg/kg)
5xVCR i.p.,
i.p.,IxVCR
(1.5 mg/kg)
min180min30 182,6,10, 14,
1822,6,
10, 14,
4times25
mg/kg i.p.,
4times"Drug"
mg/kg i.p.,
survival
(days)9.1
time
±0.1"25.2
0.625.3
±
0.718.3
±
±0.712.0
±0.011.2
i.p..4xVCR
(0.5 mg/kg)
i.p..4
mg/kg
times25
4times25
mg/kg i.p.,
of
treatment2,6,
142,6,IO,
±0.323.5
142,6,10,
1.043.3
±
i.p..4xVCR
(0.5 mg/kg)
min30
142, 10,
1.924.0
±
i.p..1
(1.5 mg/kg)
2)VCR
x (Day
i.p..Ix
(1.5 mg/kg)
(Day 1)Interval
min24
142,
6, 10,
0.733.7
±
hrDays
6, 10, 14Mean
±0.8Cures0000003/901/7
"Mean ±S.E.
sone alone (175 mg/kg, 5 times) did not prolong survival
beyond control mice with tumor. The control mean survival
of mice receiving only MTX at 25 mg/kg every 4 days for 5
doses was 25.2 days. When hydrocortisone (175 mg/kg)
preceded MTX, by 60 min, no change in survival was noted.
However, when treatment with MTX and hydrocortisone
was separated by 180 min, the mean survival time decreased
by 7 days. The difference between mean survival for 180 min
and the MTX control was statistically significant (p <
0.01).
The Vinca alkaloids are unique in their ability to enhance
transport of MTX in the L1210 leukemia cell in vitro. In
vivo experiments to evaluate the effect of VCR on MTX
antitumor activity can also be seen in Table 3. Control
survival of 9.1 days was prolonged only slightly by VCR at
1.5 mg/kg given i.p. on Day 1 or 0.5 mg/kg i.p. every 4
days, 4 times. MTX treatment with 25 mg/kg every 4 days,
4 times, increased mean survival to 23.5 days and resulted in
no long-term survivors (defined as animals surviving greater
than 90 days). VCR given at 0.5 mg/kg i.p. 30 min prior to
MTX markedly increased mean survival to 43.3 days with
30% cures. Even giving the VCR as a single dose (1.5
mg/kg) 24 hr before the 1st dose of MTX increased survival
to 33.7 days with approximately 15%cures. However, when
a single dose of 1.5 nUgof VCR per kg was given 30 min
prior to MTX (25 mg/kg every 4 days, 4 times) on Day 2
only, no increase in survival was noted.
Additional in vivo experiments involved determining the
number of viable ascites cells per mouse following i.p.
L1210 tumor inoculation. The results of these quantitative
1674
ascites experiments can be seen in Chart 5 for i.-asparaginase or VCR given prior to MTX. Tumor cells were given
i.p. on Day 0 and MTX was given i.p. on Day 3.
L-Asparaginase (3500 units/kg) or VCR (1.5 mg/kg),
when given alone to L1210-bearing mice, resulted in no
change in the number of ascites cells per mouse when
compared to control mice inoculated with tumor cells.
When given 24 hr before MTX, VCR (1.5 mg/kg) caused a
substantial curve shift to the right of the L1210 plus MTX
curve illustrating its synergistic effect with MTX in the
L1210 tumor system. Giving the VCR only 30 min before
MTX resulted in a reduced synergistic effect.
Giving the L-asparaginase (3500 units/kg) i.p. 30 min
before MTX resulted in slight antagonism of MTX antitumor activity. However, when the interval between MTX and
L-asparaginase was extended to 3 hr (Chart 5) more marked
antagonism was seen. Quantitative ascites experiments with
175 mg of hydrocortisone per kg given 30 min prior to 2 mg
of MTX per kg resulted in slight antagonism of MTX
antitumor activity (not shown in Chart 5).
The survival data presented and the killing effect on the
number of ascites cells per mouse seem to support the
conclusions that MTX transport inhibitors can antagonize
the antitumor activity of MTX in the L1210 leukemia
system and the Vinca alkaloids, transport enhancers, can
act synergistically with MTX to improve its antitumor
activity. However, the time course of effects in these
experiments indicates that factors other than transport
effects may be involved in producing changes in MTX
antitumor activity.
CANCER
RESEARCH
VOL. 33
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MTX Interactions with Other Drugs
IO1
ouT IO8
o
o
UJ
05
O
ir
ÃœJ
o.
_
UJ
u
Õ3
*
o
co
/
'
i
I
/
J
s
¡
•¿LI2IO Control
o
LI2IO»MTX»L-osparoginase (3hrs)
A LI2IO +MTX
D LI2IO +MTX +VCR(30min)
A LI2IO+MTX +VCR(24hr)
i
i
4
i
6
DAYS POST
i
i
i
8
TUMOR (LI2IO)
i
i
IO
12
IMPLANTATION
Chart 5. Effect of L-asparaginase and VCR on MTX
inhibition of
LI2IO murine leukemia cell proliferation in vivo. Each point representsthe
average of 4 mice. Doses: MTX, 15 mg/kg: VCR, 1.5 mg/kg, and
L-asparaginase, 3500 units/kg.
DISCUSSION
MTX transport in the LI210 murine leukemia cell has
been shown to be an active, carrier-mediated uphill process
(8). Studies have shown that MTX tightly binds DHFR,
with depletion of folate coenzymes (14, 15), cessation of
thymidylate synthesis (2, 20), and ultimately disruption of
DNA synthesis. Clearly, MTX must enter the cell to have
an antitumor effect. In support of this fact, it has been
shown that inability to transport MTX into a cell, in certain
animal tumor systems, is associated with resistance to this
agent (5, 18). This information has prompted our examina
tion of the effects of other "drugs" on MTX transport.
Drug-drug interactions have become increasingly impor
tant factors affecting the therapeutic response of a variety of
diseases to drug therapy. Such examples as the antagonism
between barbiturates and the anticoagulant effects of the
JULY
warfarin compounds and the effect of allopurinol on
6-mercaptopurine metabolism are recognized in the daily
use of these agents. For MTX, recent work has suggested
that drug-drug interactions may affect its antitumor activ
ity. Capizzi ei al. (3) reported the antagonism of the antineoplastic effect of MTX by L-asparaginase in the L5178Y
murine leukemia system both in vitro and in vivo. Most
recently, working with the LSI78Y murine leukemia sys
tem, Nahas and Capizzi (13) have shown a marked de
crease in cellular uptake of MTX in vitro 24 hr following
i.p. injection of 250 units of i.-asparaginase per kg.
The results of the present study show that hydrocortisone,
methylprednisolone, cephalothin, and L-asparaginase are all
inhibitors of MTX transport in the L1210 murine leukemia
cell in vitro. This inhibition is dose related and competitive
for hydrocortisone. In addition, for hydrocortisone and
L-asparaginase, antagonism of MTX antitumor activity has
been demonstrated in vivo.
The relationship between transport inhibition and antago
nism of MTX antineoplastic activity by hydrocortisone in
the L1210 system is yet to be fully explained. Since
hydrocortisone does not prevent the eventual saturation of
the DHFR binding sites in the L1210 cells, it would appear
that its ability to antagonize the antineoplastic activity of
MTX would be minimal. This conclusion is supported by
the quantitative ascites data. However, in vivo survival
experiments, using multiple doses of hydrocortisone and
MTX, showed much greater antagonism. This suggests
that, although transport inhibition may play a role in the
antagonism of MTX antineoplastic activity, other factors
are probably of greater importance.
The potency of L-asparaginase as a transport inhibitor
does not reflect its antagonistic potential. Although it is a
relatively weak transport inhibitor in the L1210 system,
quantitative ascites experiments demonstrated substantial
antagonism of MTX antitumor activity when the MTX was
administered 3 hr after L-asparaginase. Lieberman et al.
(11) has shown that inhibition of protein synthesis can
protect
against
cell death
induced
by
1-0-Darabinofuranosylcytosine, a cell-cycle-specific agent similar
to MTX. Since L-asparaginase is a known inhibitor of
protein synthesis and an interval of approximately 3 hr
seems necessary to demonstrate maximum antagonism,
protein synthesis inhibition may be the mechanism of
antagonism of MTX antitumor activity. In the L1210
system, cephalothin, hydrocortisone, and L-asparaginase
appear capable of interfering with MTX antitumor activity.
The clinical significance of these observations for MTX
chemotherapy in man is yet to be determined.
Vinca alkaloids uniquely enhance MTX transport in the
L1210 leukemia system. L12IO cells preincubated with
VCR or VBL showed enhanced MTX transport in vitro.
This effect was seen with as brief as 1 min of preincubation
and repeated washing after incubation does not prevent
enhanced transport. This transport enhancement results in
substantially higher levels of "free" MTX, not bound to
DHFR, within the L1210 cell. The role of this fraction of
the MTX in antitumor activity in the L1210 system has not
been established. While VCR, at clinically achievable levels
1973
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1973 American Association for Cancer Research.
1675
R. F. Zager, S. A. Frisby, and V. T. Oliverio
in man, acts synergistically with MTX in L1210 murine
leukemia in vivo, this phenomenon is schedule dependent
and may involve interrelated factors other than drug
transport such as cell-cycle kinetics (19) and the duration of
effective drug levels. This was apparent when no increased
survival was noted in L1210 leukemia-bearing mice given
VCR (1.5 mg/kg) 30 min prior to MTX (25 mg/kg every 4
days, 4 times) on Day 2 only. This suggests that the
enhancement of transport of MTX occurring on Day 2 only
is not sufficient to give increased survival.
This study has described several commonly used "drugs"
with an inhibitory effect on MTX transport in the L1210
leukemia system. We have also shown that they possess the
ability to antagonize the antitumor activity of MTX in vivo.
However, the relationship between these 2 phenomena is not
established and further work, particularly in human malig
nant cell lines, is needed before the possible clinical
significance of these interactions can be fully appreciated.
The enhancing effect of the Vinca alkaloids on MTX
transport and antitumor activity in the L1210 system may
be of clinical significance in the concomitant use of drugs in
cancer chemotherapy. It is envisioned that enhanced thera
peutic effect of MTX in certain tumor systems might be
achieved through a drug administration schedule that takes
advantage of the transport-enhancing effect. In particular,
this technique might be useful in the therapy of malignant
cell lines resistant by virtue of diminished MTX transport.
Appropriate drug scheduling may take advantage of the
transport-enhancing effect and the cell synchronization
effect of the Vinca alkaloids to obtain maximum therapeutic
effect from MTX.
5. Fischer, G. A. Defective Transport of Amethopterin (Methotrexate) as
a Mechanism of Resistance to the Antimetabolite in L5178Y Leu
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Counting of Plasma and Tissues. Intern. J. Appi. Radiation Isotopes.
S.-95 97, I960.
8. Goldman, I. D., Lichtenstein, N. S., and Oliverio, V. T. Carriermediated Transport of the Folie Acid Analogue, Methotrexate, in the
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CANCER RESEARCH
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VOL. 33
The Effects of Antibiotics and Cancer Chemotherapeutic Agents
on the Cellular Transport and Antitumor Activity of Methotrexate
in L1210 Murine Leukemia
Robert F. Zager, Shayla A. Frisby and Vincent T. Oliverio
Cancer Res 1973;33:1670-1676.
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