Download Overcoming of Vincristine Resistance in P388

[CANCER RESEARCH 41, 1967-1972,
0008-5472/81
70041-OOOOS02.00
May 1981]
Overcoming of Vincristine Resistance in P388 Leukemia in Vivo and in
Vitro through Enhanced Cytotoxicity of Vincristine and Vinblastine
by Verapamil1
Takashi Tsuruo,2 Harumi lida, Shigeru Tsukagoshi, and Yoshio Sakurai
Cancer Chemotherapy
Center, Japanese Foundation for Cancer Research, Toshima-ku, Tokyo I 70. Japan
ABSTRACT
A noncytotoxic dose of verapamil, a coronary vasodilator,
enhances the cytotoxicity of Vincristine (VCR) and vinblastine
in P388 leukemia and its VCR-resistant subline, P388/VCR.
When 2.2 to 6.6 JUMverapamil was added along with VCR to
the P388/VCR culture in vitro, VCR resistance was completely
overcome. Verapamil in doses of 50 to 100 mg/kg adminis
tered daily for 10 days with VCR also enhances the chemotherapeutic effect of VCR in P388- and, especially, P388/VCRbearing mice. When approximately 3 times the amount of VCR
was given to a P388/VCR bearer as compared to a P388
bearer, VCR resistance was almost completely overcome in
vivo with 50 to 100-mg/kg doses of verapamil. The amount of
VCR incorporated into P388 cells was larger than that in P388/
VCR cells. Verapamil (6.6 ¡J.M)
enhanced the cellular level of
VCR in P388 cells 2-fold and enhanced the level of VCR in
P388/VCR cells 10-fold. The amount of VCR in P388/VCR
cells reached the same level as that found in P388 cells. The
overcoming of VCR resistance in vivo and in vitro could be
explained by the effective accumulation of VCR by verapamil
in P388/VCR cells mediated by the inhibition of a VCR efflux
function of the cells, a mechanism which remains to be solved.
INTRODUCTION
The Vinca alkaloids,
VCR3 and VLB, isolated from Vinca
rosea L., are commonly used as chemotherapeutic agents in
the treatment of cancer (5, 25). Although the mechanism of
action of the drugs has not been clearly elucidated, the major
antitumor effect of these agents appears to be related to their
action on tubulin and microtubules (19, 25). Microtubules and
microfilaments, components of the cytoskeletal structure, con
nect either directly or indirectly to macromolecules in the
plasma membrane and participate in the regulation of a number
of membrane-associated cellular events (1, 12, 21 ).
We have examined the effects of a series of membraneinteracting agents on the cytotoxicity of Vinca alkaloids against
cultured cells. We have been exploring the possibility that the
membrane-modifying
agent might affect the function(s) of mi
crotubules or alter the transport function of the drugs through
the plasma membrane, resulting in an enhanced cytotoxicity of
' This work was supported by Grant-in-Aid for Cancer Research 40101 7 from
the Ministry of Education, Science, and Culture. Japan.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: VCR, Vincristine; VLB, vinblastine; P388/VCR.
P388 leukemic cells resistant to VCR; T/C. mean survival time of treated group
of mice divided by mean survival time of control group; PBS. phosphate-buffered
saline consisting of 0.02 M sodium phosphate-0.15
M NaCI, pH 7.4; ICso,
concentration of drug required for 50% inhibition of cell growth.
Received August 8, 198*0; accepted January 22, 1981.
Vinca alkaloids for tumor cells. In this communication, we have
examined the effect of verapamil on the cytotoxicity of VCR
and VLB for P388 leukemia and its VCR-resistant subline
(P388/VCR) in vitro and in vivo. Verapamil is a clinically used
coronary vasodilator (10, 11 ). The primary target of verapamil
is presumed to be the membranes because the drug has
lipophilic side chains [(—OCH3)4] (2). A well-known action of
verapamil is its inhibition of the slow channel of Ca2+ transport
across the membranes (10, 15, 16), although the mechanism
of this action has not been clearly elucidated. Another note
worthy effect of verapamil is its action on secretions. Verapamil
blocks the release of oxytocin and vasopressin from the de
polarized neurohypophysis (8, 22) and that of insulin from
excited /S-cells in the islets of Langerhans (7, 17). The drug
also suppresses the secretion of adrenocorticotropin,
growth
hormone, and thyroid-stimulating
hormone (9). We found in
this study that verapamil at a nontoxic dose inhibited the efflux
of cellular VCR and enhanced the cytotoxicity of Vinca alkaloids
against P388 and its VCR-resistant subline. VCR resistance in
P388 leukemia has been overcome in vitro and in vivo.
MATERIALS
AND METHODS
Drugs. VCR sulfate and VLB sulfate formulated for clinical
use were obtained from Shionogi and Co., Ltd., Osaka, Japan,
and [3H]VCR sulfate (2.8 Ci/mmol) was purchased from the
Radiochemical Centre, Amersham, Buckinghamshire, England.
Verapamil was kindly supplied by the Eisai Co., Ltd., Tokyo,
Japan.
Animals and Tumors. Adult female BALB/c x DBA/2Cr F,
(hereafter called CD2Fi) mice weighing 20 to 23 g were used
in experiments; DBA/2Cr mice were the carriers of P388
leukemia and its VCR-resistant subline. CD2F, and DBA/2Cr
mice and P388 leukemic cells were supplied by Simonsen
Laboratories, Inc., Gilroy, Calif., under the auspices of the
National Cancer Institute, NIH, Bethesda, Md. P388/VCR was
kindly supplied by the Mammalian Genetics and Animal Pro
duction Section, Division of Cancer Treatment, National Cancer
Institute, NIH, Bethesda, Md.
Evaluation of Antitumor Activity. One-tenth ml of diluted
ascites fluid containing 106 P388 or P388/VCR cells was
transplanted i.p. into CD2F, mice. Verapamil and VCR or VLB
were dissolved in 0.9% NaCI solution. Except as otherwise
indicated, both drugs were mixed, and the mixture was admin
istered at a constant rate of 0.01 ml/g body weight i.p. daily
for 10 days starting from the day after the tumor inoculation.
Doses of verapamil and VCR (or VLB) were in the range of 50
to 125 mg/kg and 1 to 200 jug/kg, respectively. Antitumor
activity was expressed by: (a) T/C; (b) at each dosage of VCR
and VLB, the mean survival time of the treated group divided
MAY 1981
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1967
T. Tsuruo et al.
by the mean survival time of the group of mice treated with
VCR or VLB alone. Five mice were used for each experimental
group.
Cell Culture and Drug Treatment. P388 and P388/VCR
ascites cells were harvested from the peritoneal cavity of each
tumor-bearing DBA/2Cr mouse. The cells were maintained in
plastic dishes (Corning Glass Works, Corning, N. Y.) in Roswell
Park Memorial Institute Medium 1640 supplemented with 10%
fetal calf serum (Grand Island Biological Co., Grand Island, N.
Y.), 20 UM 2-mercaptoethanol, and kanamycin (100 jug/ml) (3).
The cultures were incubated at 37°in a humidified atmosphere
of 5% CO2. The cells were subcultured twice and then used for
experiments. As a rule, the cells were kept continuously in
culture for less than 3 weeks, and there was essentially no
change in drug sensitivity and VCR resistance during that
period. Under these conditions, the doubling time for P388 and
P388/VCR cells was 17 and 25 hr, respectively. For the drug
treatment experiment, culture medium (2 ml) containing 1 x
10" P388 and 1.5 x 10" P388/VCR cells/ml of the medium,
respectively, was transferred to Falcon No. 2054 culture tubes
(Falcon Plastics, Oxnard, Calif.). Two tubes were used for each
drug concentration. The tubes were incubated at 37° in a
humidified atmosphere of 5% CO2. Twenty-four hr later, the
cell densities of P388 and P388/VCR cells reached approxi
mately 2.25 x 10" cells/ml of medium. Verapamil and VCR or
verapamil was added to the mixture at a final concentration of
2.2 or 6.6 ¡J.M.
The mixture was incubated for 15 min at 37°.
The extent of binding of [3H]VCR to tubulin was then determined
by the filter assay technique (18, 20), whereby the incubate
was filtered through a Whatman DE81 filter, followed by a
washing with 0.01 M sodium phosphate buffer, pH 6.5, con
taining 0.01 M MgCI2. The radioactivity retained on the filter
was counted in 10 ml Econofluor (New England Nuclear) in a
Beckman LS 7500 scintillation system.
RESULTS
Enhanced Cytotoxicities of VCR and VLB in P388 and
P388/VCR Cells by Verapamil. Both P388 and P388/VCR
cells showed the same sensitivity against verapamil. At vera
pamil concentrations up to 6.6 p.M, no growth inhibition was
observed for both cells; at 23 JUM,only marginal inhibition
(approximately 3%) was noted. The IC50 of verapamil for both
cells was 50.5 /ÕM.Approximately 70 and 100% inhibition
occurred at 66 and 230 JUMverapamil, respectively.
The sensitivities of P388 and P388/VCR cells to VCR and
VLB and the effect of verapamil on the sensitivity are illustrated
in Chart 1. P388/VCR cells were resistant to VCR and also to
VLB. The index of resistance of P388/VCR cells to VCR was
31, and the IC50's of VCR for P388 and P388/VCR were 1.4
VLB dissolved in PBS were added successively to the culture,
and the cells were cultivated further for another 48 hr. Cells
were then counted with a Coulter counter (28). The cytotoxic
activity of VCR or VLB in the presence or absence of verapamil
was measured by determining the IC50 which was obtained by
plotting the logarithm of the drug concentration versus the
growth rate (percentage of control) of the treated cells (28).
The initial cell number was subtracted in the calculation.
Cellular Uptake and Retention of [3H]VCR. P388 or P388/
VCR cells (1.5 x 106) in the flasks containing 50 ml of the
medium with 20 HIM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (Grand Island Biological Co.) were incu
bated at 37° in the presence of [3H]VCR (10 nw; specific
and 44 nM, respectively, while the index of resistance of P388/
VCR cells to VLB was 7 and the IC50's of VLB for P388 and
activity, 2.8 Ci/mmol) with or without verapamil (6.6 fiM, cor
responding to 3 jug/ml of medium). At various time intervals,
the culture was mixed well, and two 1-ml and two 5-ml aliquots
were withdrawn. The cells were enumerated using the 1-ml
aliquots. The 5-ml aliquots were each mixed with ice-chilled
PBS (5 ml) containing 2 x 106 P388 cells, and the mixture was
centrifuged at 300 x g for 5 min at 4°.The supernatant fluid
0.36 nM, respectively.
The same phenomenon occurred with VLB when verapamil
was added with VLB to the culture (Chart 1B). Verapamil (2.2
juM) rendered the P388/VCR cells sensitive to VLB just as with
P388 cells (IC50 was 1.6 nM for P388 and 1.7 nM for P388/
VCR cells), and at 6.6 /¿M
verapamil the IC60's of VLB for P388
was discarded by décantation, and the pelleted cells were
suspended with 10 ml cold PBS and centrifuged at 500 x g
for 5 min. The pelleted cells were lysed overnight with 1 ml of
Protosol (New England Nuclear, Boston, Mass.) and trans
ferred to a scintillation vial containing 10 ml of Econofluor (New
England Nuclear), and the radioactivity was counted in a Beckman LS 7500 liquid scintillation system equipped with auto
matic quench compensation. Counting efficiency was 54 to
55%.
Binding Assay of VCR to Tubulin. Purified tubulin, prepared
from porcine brain by the method of Shelanski ef a/. (24), was
a gift from Dr. H. Sakai, University of Tokyo. Tubulin (10 jug)
was mixed with 0.25 to 2.0 nmol of [3H]VCR (specific activity,
1 Ci/mmol) in 1 ml of 0.01 M sodium phosphate buffer, pH
6.5, containing 0.1 rriM GTP (18, 20). When the effect of
verapamil on the binding of VCR to tubulin was examined,
1968
P388/VCR cells were 3.0 and 21 nM, respectively. Verapamil
at a nontoxic dose of 2.2 and 6.6 fiM greatly enhanced the
cytotoxicity of VCR for P388 cells and, especially, for P388/
VCR cells (Chart 1/4). When verapamil was added at a final
concentration of 2.2 fiM to P388/VCR cell cultures, the IC50of
VCR shifted from 44 to 1.3 nM. This value was almost the same
as the IC5o (1.4 nM) of VCR for P388 cells in the absence of
verapamil. In the presence of verapamil (2.2 ¿IM),the IC50 of
VCR for P388 cells was 0.48 nM. At 6.6 JUMverapamil, almost
the same growth inhibition occurred in both P388 and P388/
VCR cells and the IC50's of VCR for these cells were 0.37 and
and P388/VCR cells were 0.45 and 0.34 nM, respectively.
Thus, resistance of P388 cells against Vinca alkaloid could be
completely overcome at a nontoxic dose of verapamil in vitro.
Combined Effect of Vinca Alkaloid and Verapamil on P388and P388/VCR-bearing Mice. VCR administered daily for 10
days starting from Day 1 increased the life span of P388
leukemia-bearing mice. T/C values were 102, 132, and 146%,
respectively, at VCR dosages of 1, 10, and 30 ftg/kg, respec
tively (Table 1A). Verapamil administered at 50 to 100 mg/kg
with VCR further increased the life span (10 to 20%) of the
tumor bearer, although verapamil alone at 100 mg/kg showed
no therapeutic effect. Verapamil at 125 mg/kg administered
10 times was toxic.
VCR given according to the schedule above showed no
therapeutic effect against P388/VCR-bearing
mice except for
the dosage of 200 /¿g/kg where a slightly higher T/C value
(107%) was obtained (Table 1B). However, verapamil given 10
CANCER
RESEARCH
VOL. 41
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Effect of Verapamil on VCR Cytotoxicity
Table 1
Effect of verapamil on antitumor activity of VCR in P388- and P388/VCRbearing mice
Each group of 5 CD2F, mice was given i.p. implants of 106 cells of P388 or
100
so
P388/VCR leukemia on Day 0, and drugs were given i.p. daily from Days 1 to
10, except for Group C. in which drugs were given from Days 1 to 5.
60
dosageA.
time
(days)10.0
Drug and
miceControlVerapamil
P388-bearing
0.9610.0
±
40
I
I
0.1
1
0.814.6
±
0.916.8
±
1.916.4
±
2.116.3
±
2.213.2
±
0.814.8
±
0.815.0
±
0.714.4
±
0.510.2
±
0.412.0
±
1.911.4
±
0.912.2
±
1.111.0
±
mg/kg)VCR (100
/ig/kg)+
(30
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)VCR(10/ig/kg)+
Verapamil (50
20
10
100
Concentration
of vincristine( nM )
100
(%)a100115a1121121001
(%)100100146C168°164C163C13
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)VCR
Verapamil (50
(¿g/kg)+
(1
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)B.Verapamil (50
80
miceControlVerapamil
P388/VCR-bearing
40
5
0.69.6
±
1.511.8
±
0.812.6
±
1.215.5
±
0.615.2
±
0.410.6
±
0.516.0
±
015.0 ±
0.714.2
±
1.510.8
±
1.114.2
±
0.812.8
±
1.112.8
±
1.1to ±
mg/kg)VCR (1 00
kg)+
(200 /ig/
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)VCRdOO/jg/kg)+
Verapamil (50
20
0.1
1
10
Concentration
of vinblastlne( nM )
100
Chart 1. Effects of verapamil upon growth-inhibitory actions of VCR and VLB
on P388 and P388/VCR leukemia cells. P388 and P388/VCR were seeded in
2 ml of Roswell Park Memorial Institute Medium 1640 containing 10% fetal
bovine serum, 20 UM 2-mercaptoethanol,
and kanamycin (100 fig/ml) at 1 and
1.5x10"
cells/ml of medium, respectively. Twenty-four hr later, the cell density
reached approximately 2.25 x 104 cells/ml of medium. The cells were incubated
with drugs as follows, and the cell numbers were counted 2 days after the drug
treatment. In A, P388 cells were incubated with VCR at the indicated concentra
tions in the absence {• •)or presence of verapamil at 2.2 (A
A) and
6.6 (• •)/UM, and ICso's of VCR were 1.4, 0.48, and 0.37 nM, respec
tively. P388/VCR cells were treated with VCR at the indicated concentrations in
the absence (•
-•)or presence of verapamil at 2.2 (A
A) and 6.6
(•
•)fiM, and ICso's of VCR were 44, 1.3, and 0.36 nw, respectively. In B,
P388 cells were treated with VLB at the indicated concentrations in the absence
(• •)or presence of verapamil at 2.2 (A
A) and 6.6 (• •)JIM, and
ICuo's of VLB were 3.0, 1.6, and 0.45 nM, respectively. P388/VCR cells were
treated with VLB at the indicated concentrations in the absence (•
•)or
presence of verapamil at 2.2 (A
A) and 6.6 (•
•)UM, and ICso's of VLB
were 21, 1.7, and 0.34 nM, respectively.
times with VCR significantly increased the life span of the
P388/VCR bearer. Especially notable was a 40 to 50% in
crease in life span which was observed for the P388/VCR
bearer when verapamil (75 to 100 mg/kg) was administered
with VCR (100 jug/kg). At a VCR dose of 30 jug/kg ¡nthe
P388/VCR bearer, a T/C value of 129% was obtained with a
100-mg/kg dose of verapamil. This value was less than that
(146%) obtained in the P388 bearer treated with VCR alone at
30 /¿g/kg. However, VCR (100 jug/kg) administered with ver
apamil (75 to 100 mg/kg) to the P388/VCR bearer increased
the life span of the mice, and T/C values of 136 to 145% were
obtained. Because these values are close to that (146%) ob
tained ¡nthe P388 bearer treated with VCR alone at 30 /ig/kg,
it can be said that VCR resistance could be almost completely
overcome in the P388/VCR bearer when approximately triple
amounts of VCR were given with verapamil. T/C percentage
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)VCR
Verapamil (50
/kg)+
(30 fig
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)C.Verapamil (50
1ControlVerapamil
P388/VCR-bearing
mice (Therapy Days
mg/kg)VCR (1 25
/kg)+
(200 jig
mg/kg)+
Verapamil (125
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)VCR
Verapamil (50
fig/kg)+
(100
mg/kg)+
Verapamil
mg/kg)+
Verapamil
mg/kg)+
Verapamil
mg/kg)VCR
Verapamil
/ig/kg)+
(30
mg/kg)+
Verapamil
mg/kg)+
Verapamil
mg/kg)+
Verapamil
Verapamil
(125
(100
(75
(50
(125
(100
(75
(50 mg/kg)Survival
5)11.6
1.511.2
±
0.411.0
±
08.4 ±
4.31 ±
0.415.5
1.2 ±
1.011.6
±
2.211.0
±
1.915.5
±
1.314.6
±
0.514.2
±
0.413.0
±
1.411.0
±
0.714.0
±
014.0 ±
013.6 ±
0.912.8
±
±1.1T/C
T/V, at each VCR dosage, the mean survival time of the treated group
divided by the mean survival time of the group of mice treated with VCR alone.
b Mean ±S.D.
c Statistically significant (p < 0.05) by Student's ( test as compared with that
of the control experiment.
a Statistically significant (p < 0.05) by Student's ( test as compared with that
of mice treated with VCR alone at each dosage of VCR.
value (132%) of the P388 bearer treated with VCR alone at 10
jug/kg was similar to that (129%) obtained in the P388/VCR
bearer which was treated with VCR at triple amounts (30 fig/
kg) and verapamil (100 mg/kg).
A significant increase of T/C value was also observed in
P388/VCR-bearing
mice when VCR and verapamil were given
daily for 5 days (Table 1C). The dose of verapamil could be
increased to 125 mg/kg without manifestation of toxicity.
However, VCR (200 jug/kg) with verapamil (125 mg/kg) was
toxic. A significant effect of verapamil was observed with VCR
(200 /ig/kg) plus verapamil (75 mg/kg), and at 100- and 30-
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1969
T. Tsuruo et al.
/ig/kg doses of VCR with verapamil (75 to 125 mg/kg). How
ever, the effects were less than those obtained in the experi
ments with drug treatment for 10 successive days.
When VLB was used instead of VCR, a similar enhancement
of antitumor activity of VLB occurred (Table 2). Enhancement
in P388-bearing mice was small as has been observed in the
experiment with VCR (Table 2A). However, VLB (100 /xg/kg)
plus verapamil (50 to 100 mg/kg) increased the life span of
the P388/VCR bearer by approximately 30% when compared
to the group of mice treated with VLB alone (Table 2B). Al
though this value is less than that obtained in the experiment
with VCR, the results indicated that VCR resistance can also
be partially overcome by VLB and verapamil in vivo.
Cellular Uptake of VCR and the Effect of Verapamil. Cel
lular uptake of VCR was examined in the presence of 10 nM
[3H]VCR. The most prominent effect of verapamil on the cytotoxicity of VCR against P388/VCR cells has been obtained at
10 nM VCR as is shown in Chart 1. More than 98% of P388
and P388/VCR cells excluded trypan blue after treatment of
the cells with 10 nM VCR and 6.6 juM verapamil for 5 hr.
Furthermore, treatment of the cells with 6.6 fiM verapamil did
not change cellular uptake rates of a-aminoisobutyric acid and
2-deoxyglucose. These results might indicate that the plasma
membrane and membrane permeability of the cells were kept
intact during the drug treatment. Uptake of [3H]VCR into cul-
tured P388 cells increased with time under the conditions of
constant drug exposure (Chart 2). Approximately 0.85 pmol of
VCR was found at 5 hr in 106 P388 cells, while the amount of
VCR in P388/VCR cells was much smaller and the level almost
reached a plateau (0.1 pmol/106 cells) after 1 hr of incubation;
only a marginal increase occurred thereafter. The mechanism
of resistance could be explained by this phenomenon. Vera
pamil added to the culture at 6.6 ¿IMgreatly increased the
amount of cellular VCR in both P388 and P388/VCR cells.
Approximately twice the amount of VCR was found in P388
cells treated with verapamil. While almost a 10-fold accumula
tion of VCR occurred in verapamil-treated P388/VCR cells
during 3 to 5 hr of incubation, the amount of VCR reached a
slightly higher level than that in P388 cells. Enhanced cytotoxicity of VCR in P388 and P388/VCR cells by verapamil and
the overcoming of VCR resistance in P388/VCR cells in vivo
and in vitro by verapamil could be explained by this phenom
enon.
The enhanced accumulation of VCR in verapamil-treated
cells could be explained by the following possibilities: (a)
verapamil enhances the affinity of VCR for tubulin in the cells;
(o) verapamil enhances the influx of VCR into cells; (c) vera
pamil inhibits the efflux of intracellular VCR.
Effect of Verapamil on the Binding of [3H]VCR to Tubulin.
The binding of [3H]VCR to tubulin increased with the amount of
[3H]VCR added to the reaction mixture (Chart 3). Verapamil did
not show any significant effect on the binding of [3H]VCR to
Table 2
Effect of verapamil on antitumor activity of VLB in P388- and P388/VCRbearing mice
tubulin, indicating that verapamil does not modify the affinity of
VCR for tubulin.
Each group of 5 CD2F, mice was given i.p. implants of 106 cells of P388 or
Effect of Verapamil on the Transport of VCR in P388/VCR
P388/VCR leukemia on Day 0. and drugs were give i.p. daily from Days 1 to 10. Cells. Verapamil seemed not to enhance the influx of VCR into
time
P388/VCR cells, inasmuch as the pretreatment of the cells
(%)a100109d109d10310010511010210010410212110092108"107"100132d128d123rf100128rf121a114"
dosageA. Drug and
(days)10.0
(%)10088138°150°150C142C122C128°134C124C104108106126100102127C117137C136C103136C132C127°9
with verapamil had no effect on the cellular accumulation of
miceControlVerapamil
P388-bearing
VCR (Chart 4). The efflux of intracellular VCR from P388/VCR
Ob8.8
±
cells, however, was significantly inhibited by verapamil as
mg/kg)VLB(100
2.013.8
±
ng/kg)+
(30
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)VLBOOfig/kg)+
Verapamil (50
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)VLBd
Verapamil (50
Mg/kg)+
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)B.Verapamil (50
0.515.0
±
0.715.0
±
0.714.2
±
0.812.2
±
0.412.8
±
1.013.4
0.512.4
0.510.4
0.910.8
0.410.6
0.512.6
3.611.8
±
miceControlVerapamil
P388/VCR-bearing
0.812.0
±
1.215.0
±
013.8±
5.416.2
±
0.416.0
±
0.712.2
±
1.116.0
±
1.015.6
±
0.915.0
±
1.011.2
±
0.814.3
±
0.513.6
±
2.112.8
±
mg/kg)Survival
±1.1T/C
mg/kg)VLB(100
,,g/kg)+
(200
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)VLB(100/ig/kg)+
Verapamil (50
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
mg/kg)VLB
Verapamil (50
fig/kg)+
(30
mg/kg)+
Verapamil (100
mg/kg)+
Verapamil (75
Verapamil (50
T/V, at each VLB dosage, the mean survival time of the treated group
divided by the mean survival time of the group of mice treated with VLB alone.
Mean ±S.D.
0 Statistically significant (p < 0.05) by Student's f test as compared with that
Chart 2. Effects of verapamil on the uptake of [3H]VCR by P388 and P388/
VCR leukemic cells. P388 cells (1.5 x 106) were incubated in 50 ml of Roswell
Park Memorial Institute Medium 1640 containing 10% fetal bovine serum and 20
mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid buffer at 37°with 10 nw
[3H]VCR (specific activity, 2.8 Ci/mmol) in the absence (• •)or presence of
of the control experiment.
Statistically significant ( p < 0.05) by Student's f test as compared with that
verapamil at 6.6 JIM(O
O). P388/VCR cells were also incubated with VCR as
above in the absence (•
•)or presence of verapamil at 6.6 /IM (O
O).
At time intervals, aliquots of 5 ml were removed, and the amounts of [3H]VCR
incorporated into the cells were determined as described in "Materials and
Methods." Cells were counted with 1-ml aliquots. Each point is the mean of
of mice treated with VLB alone at each dosage of VLB.
duplicate determinations.
1970
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Effect of Verapamil on VCR Cytotoxicity
-
i.o
0.5
0.5
Vincristine
Chart 3. Effect of verapamil
1.0
1.5
2.0
( nmol )
on the binding of [3H]VCR to tubulin. Purified
tubulin (10 fig) from porcine brain was incubated in 1 ml of 0.01 M sodium
phosphate buffer, pH 6.5. containing 0.1 mM GTP, with graded concentrations
of [3H]VCR (specific activity. 1 Ci/mmol) in the absence (•)or presence of
verapamil at a final concentration of 2.2 (A) or 6.6 (•)/IM. The mixture was
incubated for 15 min at 37°.The extent of binding of [3H]VCR to tubulin was then
determined by the filter assay technique (18, 20) as described in 'Materials and
Methods."
T,
.?
2.0
-'
Chart 5. Effect of verapamil on the release of [3H]VCR from P388/VCR
cells.
As described in the legend to Chart 4, the culture mixture (200 ml) containing 1
X 10' cells was incubated at 37°with 10 nM [3H]VCR (specific activity. 2.8 Ci/
mmol) in the presence of 6.6 /IM verapamil. Three hr later, the mixture was
centrifuged at 80 x g for 10 min at 5°,and the precipitated cells were suspended
in the above culture mixture at a cell density of 3 x 10* cells/ml of culture
mixture. Four 50-ml mixtures (Mixtures A to D) were prepared. In Mixtures B and
D. 6.6 JIM verapamil was added. Mixtures A and B were incubated at 37°, and
Mixtures C and D were incubated at 25°. At time intervals and as described in
the legend to Chart 4, the amounts of [3H)VCR retained in the cells were
determined in Mixture A (• •),in Mixture B (O
O), in Mixture C
(•
•),and in Mixture D (O
O). Each point is the mean of duplicate
determinations.
-
intracellular VCR and its inhibition by verapamil; the velocity of
VCR release and extent of inhibition by verapamil were almost
the same, respectively, as observed above. The velocity of
efflux of intracellular VCR decreased significantly when the
efflux was measured at 25°. Approximately 58, 15, and 6%,
1.0 -
respectively, of the initial amount of VCR remained in the cells
at 1, 3, and 5 hr after incubation at 25°. Verapamil also
inhibited the VCR efflux at 25°. Approximately, 76, 67, and
64% respectively, of the initial amount of VCR still remained in
the cells at 1, 3, and 5 hr after incubation at 25°with verapamil.
-
0'
Chart 4. Effect of pretreatment of P388/VCR cells with verapamil on the
cellular uptake of [3H]VCR. Culture mixtures (100 ml) as described in the legend
to Chart 2, were prepared and divided into 50-ml aliquots (Mixtures A and B).
Each contained 1.5 x 106 P388/VCR cells. Mixture A was incubated at 37° in
the presence of verapamil at 6.6 JUM,and Mixture B was incubated without
verapamil. Three hr later, 10 nM [3H]VCR (specific activity, 2.8 Ci/mmol) was
added to Mixture A, 10 nM [3H]VCR (specific activity, 2.8 Ci/mmol) and verapamil
(final concentration.
6.6 /ÃŒM)
were added to Mixture B, and the cells were
cultivated. At time intervals, cellular uptake of I3H ]VCR was determined with
Mixture A (•)and Mixture B (O) as described
point is the mean of duplicate determinations.
in the legend to Chart 2. Each
described below (Chart 5). The cells were preincubated with
[3H]VCR and verapamil for 3 hr, and then the cells were further
incubated at 37°or 25°with or without verapamil. At 1 hr after
incubation at 37°, about 95% of intracellular VCR was lost
from the cells incubated without verapamil; while more than
70% of the drug was retained in the cells when the cells were
incubated with verapamil. At 3 and 5 hr after incubation with
verapamil, approximately 45 and 30%, respectively, of the
initial amount of VCR still remained in the cells, while more than
99% of intracellular VCR was lost from the cells when the cells
were incubated without verapamil. Unlabeled VCR (10 nM)
added to the efflux bath had no effect on the release of
From these results, we can state that the higher accumulation
of VCR in P388/VCR cells by verapamil could occur through
an inhibition of the efflux mechanism of VCR by verapamil. A
similar inhibition by verapamil was also observed for P388
cells.
DISCUSSION
Verapamil has enhanced the cytotoxicity of VCR in both
P388 and P388/VCR cells and could completely overcome
VCR resistance in vitro. Verapamil also enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing
mice, in which
VCR resistance could be partially overcome by verapamil.
Especially when approximately 3 times the amount of VCR was
given in P388/VCR-bearing
mice along with verapamil, VCR
resistance was almost completely overcome in vivo. Using 6.6
/IM verapamil, the cellular level of VCR was enhanced to a
similar extent in both P388 and P388/VCR cells (Chart 2).
Actually, in in vitro experiments, the sensitivities of P388 and
P388/VCR to VCR were almost equal when 6.6 ¿IM
verapamil
was added to the culture (Chart 1/4). However, in in vivo
experiments, we needed approximately 3 times the amount of
VCR to obtain a similar therapeutic effect in P388- and P388/
VCR-bearing mice. A more complicated response might occur
in in vivo experiments. Among the schedules of drug adminis-
MAY 1981
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1971
f. Tsuruo et al.
tration examined, the most effective therapeutic response was
obtained when VCR (or VLB) and verapamil were given together
for 10 or 5 successive times. Administration of VCR and
verapamil on Days 1 and 5 or administration of VCR on Days
1 and 5 and verapamil on Days 1,2,3, 5, 6, and 7 to P388/
VCR bearers had no significant therapeutic effect. It is impor
tant that constant exposure of the resistant cells with both VCR
and verapamil seems to be essential to overcome resistance.
The cellular concentrations of VCR in P388/VCR cells were
3 to 5 times lower than those found in P388 cells. We observed
that the efflux of intracellular VCR occurred more rapidly for
P388/VCR cells than for P388 cells. Inaba era/. (13, 14) has
reported that the mechanism of drug resistance is the active
efflux of the intracellular drug from the resistant cells. The
possibility that verapamil alters membrane permeability could
be denied, because verapamil did not change membrane intactness as determined by the trypan blue dye exclusion test
and cellular uptake rates of a-aminoisobutyric
acid and 2deoxyglucose. Furthermore, verapamil did not change the af
finity of VCR for tubulin (Chart 3). We speculate that verapamil
inhibits the drug efflux function of the cells and thus that a very
efficient increase in drug sensitivity could be obtained in re
sistant cells. The mechanisms involved in the inhibition of drug
efflux by verapamil is not known, although it is presumably a
temperature-dependent
reaction. Oxytocin, vasopressin, insu
lin, adrenocorticotropin,
growth hormone, and thyroid-stimu
lating hormone secretions from the cells have been suppressed
by verapamil (7-9, 17, 22), although the mechanism is also
unclear at the present time. Verapamil also inhibits Ca2+ trans
fer through a slow channeling process of the membranes (10,
15, 16). Either one or both of these functions are presumably
related to the mechanism of inhibition of drug efflux from the
cells. For elucidation of the mechanism, we must examine the
effects of other Ca2+ antagonists using the present experimen
tal system and the effect of verapamil on the release of other
anticancer drugs and cellular components from the cells.
It might be possible to overcome the drug resistance practi
cally by using the approach described in this paper, if active
efflux of the drug is the cause of resistance. Such a mechanism
is widely observed in many experimental tumor cells (4, 6, 13,
14, 23, 26, 27). The application of verapamil in practical
therapy might be difficult as the drug possesses coronary
vasodilator activity. However, we can still speculate upon the
possibility of finding effective drugs which possess a stronger
inhibitory action on drug efflux with fewer side reactions than
those of verapamil. These possibilities might evolve from a
series of membrane-modifying agents such as Ca2+ antago
2. Bayer, R., Kaufman, R., and Mannhold, R. Pattern of inotropic effects of the
optical isomers. Naunyn-Schmiedeberg's
Arch. Pharmacol., 290. 49-80,
1975.
3. Broome, J. D., and Jeng, M. W. Promotion of replication in lymphoid cells by
specific thiol and disulfides/n vitro. J. Exp. Med., 738: 574-592, 1973.
4. Carlsen, S. A., Till, J. E., and Ling, V. Modulation of drug permeability in
Chinese hamster ovary cells: possible roles for phosphorylation of surface
glycoproteins. Biochim. Biophys. Acta, 467. 238-250. 1977.
5. Carter, S. K., and Livingston, R. B. Plant products in cancer chemotherapy.
Cancer Treat. Rep., 60: 1141-1156,
1976.
6. Dana, K. Active outward transport of daunomycin in resistant Ehrlich ascites
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ACKNOWLEDGMENTS
We thank the Eisai Co., Ltd., and Dr. H. Sakai, University of Tokyo, for gifts of
verapamil and purified tubulin, respectively. We are indebted to H. Bowser for
editing the manuscript.
25.
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CANCER
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VOL. 41
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Overcoming of Vincristine Resistance in P388 Leukemia in Vivo
and in Vitro through Enhanced Cytotoxicity of Vincristine and
Vinblastine by Verapamil
Takashi Tsuruo, Harumi Iida, Shigeru Tsukagoshi, et al.
Cancer Res 1981;41:1967-1972.
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