[CANCER RESEARCH 41, 1967-1972, May 1981]0008-5472/81 70041-OOOOS02.00

Overcoming of Vincristine Resistance in P388 Leukemia in Vivo and in

Vitro through Enhanced Cytotoxicity of Vincristine and Vinblastineby 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 vinblastinein P388 leukemia and its VCR-resistant subline, P388/VCR.

When 2.2 to 6.6 JUMverapamil was added along with VCR tothe P388/VCR culture in vitro, VCR resistance was completelyovercome. Verapamil in doses of 50 to 100 mg/kg administered daily for 10 days with VCR also enhances the chemo-therapeutic effect of VCR in P388- and, especially, P388/VCR-

bearing mice. When approximately 3 times the amount of VCRwas given to a P388/VCR bearer as compared to a P388bearer, VCR resistance was almost completely overcome invivo 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 ofVCR in P388 cells 2-fold and enhanced the level of VCR inP388/VCR cells 10-fold. The amount of VCR in P388/VCR

cells reached the same level as that found in P388 cells. Theovercoming of VCR resistance in vivo and in vitro could beexplained by the effective accumulation of VCR by verapamilin P388/VCR cells mediated by the inhibition of a VCR effluxfunction 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 inthe treatment of cancer (5, 25). Although the mechanism ofaction of the drugs has not been clearly elucidated, the majorantitumor effect of these agents appears to be related to theiraction on tubulin and microtubules (19, 25). Microtubules andmicrofilaments, components of the cytoskeletal structure, connect either directly or indirectly to macromolecules in theplasma membrane and participate in the regulation of a numberof membrane-associated cellular events (1, 12, 21 ).

We have examined the effects of a series of membrane-

interacting agents on the cytotoxicity of Vinca alkaloids againstcultured cells. We have been exploring the possibility that themembrane-modifying agent might affect the function(s) of microtubules or alter the transport function of the drugs throughthe 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 groupof mice divided by mean survival time of control group; PBS. phosphate-bufferedsaline 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 haveexamined the effect of verapamil on the cytotoxicity of VCRand VLB for P388 leukemia and its VCR-resistant subline(P388/VCR) in vitro and in vivo. Verapamil is a clinically usedcoronary vasodilator (10, 11 ). The primary target of verapamilis presumed to be the membranes because the drug haslipophilic side chains [(—OCH3)4] (2). A well-known action ofverapamil is its inhibition of the slow channel of Ca2+ transport

across the membranes (10, 15, 16), although the mechanismof this action has not been clearly elucidated. Another noteworthy effect of verapamil is its action on secretions. Verapamilblocks the release of oxytocin and vasopressin from the depolarized neurohypophysis (8, 22) and that of insulin fromexcited /S-cells in the islets of Langerhans (7, 17). The drug

also suppresses the secretion of adrenocorticotropin, growthhormone, and thyroid-stimulating hormone (9). We found inthis study that verapamil at a nontoxic dose inhibited the effluxof cellular VCR and enhanced the cytotoxicity of Vinca alkaloidsagainst P388 and its VCR-resistant subline. VCR resistance inP388 leukemia has been overcome in vitro and in vivo.

MATERIALS AND METHODS

Drugs. VCR sulfate and VLB sulfate formulated for clinicaluse 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 usedin experiments; DBA/2Cr mice were the carriers of P388leukemia and its VCR-resistant subline. CD2F, and DBA/2Cr

mice and P388 leukemic cells were supplied by SimonsenLaboratories, Inc., Gilroy, Calif., under the auspices of theNational Cancer Institute, NIH, Bethesda, Md. P388/VCR waskindly supplied by the Mammalian Genetics and Animal Production Section, Division of Cancer Treatment, National CancerInstitute, NIH, Bethesda, Md.

Evaluation of Antitumor Activity. One-tenth ml of dilutedascites fluid containing 106 P388 or P388/VCR cells was

transplanted i.p. into CD2F, mice. Verapamil and VCR or VLBwere dissolved in 0.9% NaCI solution. Except as otherwiseindicated, both drugs were mixed, and the mixture was administered at a constant rate of 0.01 ml/g body weight i.p. dailyfor 10 days starting from the day after the tumor inoculation.Doses of verapamil and VCR (or VLB) were in the range of 50to 125 mg/kg and 1 to 200 jug/kg, respectively. Antitumoractivity was expressed by: (a) T/C; (b) at each dosage of VCRand VLB, the mean survival time of the treated group divided

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by the mean survival time of the group of mice treated withVCR or VLB alone. Five mice were used for each experimentalgroup.

Cell Culture and Drug Treatment. P388 and P388/VCRascites cells were harvested from the peritoneal cavity of eachtumor-bearing DBA/2Cr mouse. The cells were maintained in

plastic dishes (Corning Glass Works, Corning, N. Y.) in RoswellPark Memorial Institute Medium 1640 supplemented with 10%fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.), 20 UM2-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 forexperiments. As a rule, the cells were kept continuously inculture for less than 3 weeks, and there was essentially nochange in drug sensitivity and VCR resistance during thatperiod. Under these conditions, the doubling time for P388 andP388/VCR cells was 17 and 25 hr, respectively. For the drugtreatment experiment, culture medium (2 ml) containing 1 x10" 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 eachdrug 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 approximately 2.25 x 10" cells/ml of medium. Verapamil and VCR or

VLB dissolved in PBS were added successively to the culture,and the cells were cultivated further for another 48 hr. Cellswere then counted with a Coulter counter (28). The cytotoxicactivity of VCR or VLB in the presence or absence of verapamilwas measured by determining the IC50which was obtained byplotting the logarithm of the drug concentration versus thegrowth 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-piperazineethane-

sulfonic acid buffer (Grand Island Biological Co.) were incubated at 37° in the presence of [3H]VCR (10 nw; specific

activity, 2.8 Ci/mmol) with or without verapamil (6.6 fiM, corresponding to 3 jug/ml of medium). At various time intervals,the culture was mixed well, and two 1-ml and two 5-ml aliquotswere withdrawn. The cells were enumerated using the 1-mlaliquots. The 5-ml aliquots were each mixed with ice-chilledPBS (5 ml) containing 2 x 106 P388 cells, and the mixture wascentrifuged at 300 x g for 5 min at 4°.The supernatant fluid

was discarded by décantation, and the pelleted cells weresuspended with 10 ml cold PBS and centrifuged at 500 x gfor 5 min. The pelleted cells were lysed overnight with 1 ml ofProtosol (New England Nuclear, Boston, Mass.) and transferred to a scintillation vial containing 10 ml of Econofluor (NewEngland Nuclear), and the radioactivity was counted in a Beck-

man LS 7500 liquid scintillation system equipped with automatic quench compensation. Counting efficiency was 54 to55%.

Binding Assay of VCR to Tubulin. Purified tubulin, preparedfrom porcine brain by the method of Shelanski ef a/. (24), wasa 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, pH6.5, containing 0.1 rriM GTP (18, 20). When the effect ofverapamil on the binding of VCR to tubulin was examined,

verapamil was added to the mixture at a final concentration of2.2 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 incubatewas filtered through a Whatman DE81 filter, followed by awashing with 0.01 M sodium phosphate buffer, pH 6.5, containing 0.01 M MgCI2. The radioactivity retained on the filterwas counted in 10 ml Econofluor (New England Nuclear) in aBeckman LS 7500 scintillation system.

RESULTS

Enhanced Cytotoxicities of VCR and VLB in P388 andP388/VCR Cells by Verapamil. Both P388 and P388/VCRcells showed the same sensitivity against verapamil. At verapamil concentrations up to 6.6 p.M, no growth inhibition wasobserved for both cells; at 23 JUM,only marginal inhibition(approximately 3%) was noted. The IC50 of verapamil for bothcells was 50.5 /ÕM.Approximately 70 and 100% inhibitionoccurred at 66 and 230 JUMverapamil, respectively.

The sensitivities of P388 and P388/VCR cells to VCR andVLB and the effect of verapamil on the sensitivity are illustratedin Chart 1. P388/VCR cells were resistant to VCR and also toVLB. The index of resistance of P388/VCR cells to VCR was31, and the IC50's of VCR for P388 and P388/VCR were 1.4

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

P388/VCR cells were 3.0 and 21 nM, respectively. Verapamilat a nontoxic dose of 2.2 and 6.6 fiM greatly enhanced thecytotoxicity of VCR for P388 cells and, especially, for P388/VCR cells (Chart 1/4). When verapamil was added at a finalconcentration of 2.2 fiM to P388/VCR cell cultures, the IC50ofVCR shifted from 44 to 1.3 nM. This value was almost the sameas the IC5o (1.4 nM) of VCR for P388 cells in the absence ofverapamil. In the presence of verapamil (2.2 ¿IM),the IC50 ofVCR for P388 cells was 0.48 nM. At 6.6 JUMverapamil, almostthe same growth inhibition occurred in both P388 and P388/VCR cells and the IC50's of VCR for these cells were 0.37 and

0.36 nM, respectively.The same phenomenon occurred with VLB when verapamil

was added with VLB to the culture (Chart 1B). Verapamil (2.2juM) rendered the P388/VCR cells sensitive to VLB just as withP388 cells (IC50 was 1.6 nM for P388 and 1.7 nM for P388/VCR cells), and at 6.6 /¿Mverapamil the IC60's of VLB for P388

and P388/VCR cells were 0.45 and 0.34 nM, respectively.Thus, resistance of P388 cells against Vinca alkaloid could becompletely overcome at a nontoxic dose of verapamil in vitro.

Combined Effect of Vinca Alkaloid and Verapamil on P388-and P388/VCR-bearing Mice. VCR administered daily for 10days starting from Day 1 increased the life span of P388leukemia-bearing mice. T/C values were 102, 132, and 146%,

respectively, at VCR dosages of 1, 10, and 30 ftg/kg, respectively (Table 1A). Verapamil administered at 50 to 100 mg/kgwith VCR further increased the life span (10 to 20%) of thetumor bearer, although verapamil alone at 100 mg/kg showedno therapeutic effect. Verapamil at 125 mg/kg administered10 times was toxic.

VCR given according to the schedule above showed notherapeutic 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

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100

so

60

40

20

I I0.1 1 10

Concentrationof vincristine( nM )

100

100

80

40

5 20

0.1 1 10

Concentrationof vinblastlne( nM )100

Chart 1. Effects of verapamil upon growth-inhibitory actions of VCR and VLBon P388 and P388/VCR leukemia cells. P388 and P388/VCR were seeded in2 ml of Roswell Park Memorial Institute Medium 1640 containing 10% fetalbovine serum, 20 UM 2-mercaptoethanol, and kanamycin (100 fig/ml) at 1 and1.5x10" cells/ml of medium, respectively. Twenty-four hr later, the cell densityreached 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 drugtreatment. In A, P388 cells were incubated with VCR at the indicated concentrations in the absence {• •)or presence of verapamil at 2.2 (A A) and6.6 (• •)/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 inthe 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, andICuo'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 (• •)orpresence 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 theP388/VCR bearer. Especially notable was a 40 to 50% increase in life span which was observed for the P388/VCRbearer when verapamil (75 to 100 mg/kg) was administeredwith VCR (100 jug/kg). At a VCR dose of 30 jug/kg ¡ntheP388/VCR bearer, a T/C value of 129% was obtained with a100-mg/kg dose of verapamil. This value was less than that(146%) obtained in the P388 bearer treated with VCR alone at30 /¿g/kg. However, VCR (100 jug/kg) administered with verapamil (75 to 100 mg/kg) to the P388/VCR bearer increasedthe life span of the mice, and T/C values of 136 to 145% wereobtained. Because these values are close to that (146%) obtained ¡nthe P388 bearer treated with VCR alone at 30 /ig/kg,it can be said that VCR resistance could be almost completelyovercome in the P388/VCR bearer when approximately tripleamounts of VCR were given with verapamil. T/C percentage

Table 1Effect of verapamil on antitumor activity of VCR in P388- and P388/VCR-

bearing miceEach group of 5 CD2F, mice was given i.p. implants of 106 cells of P388 or

P388/VCR leukemia on Day 0, and drugs were given i.p. daily from Days 1 to10, except for Group C. in which drugs were given from Days 1 to 5.

Drug anddosageA.

P388-bearingmiceControlVerapamil

(100mg/kg)VCR(30/ig/kg)+Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)VCR(10/ig/kg)+

Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)VCR(1(¿g/kg)+Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)B.

P388/VCR-bearingmiceControlVerapamil

(1 00mg/kg)VCR(200 /ig/kg)+Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)VCRdOO/jg/kg)+

Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)VCR(30 fig/kg)+Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)C.

P388/VCR-bearing mice (Therapy Days1ControlVerapamil

(1 25mg/kg)VCR(200 jig/kg)+Verapamil (125mg/kg)+Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)VCR(100fig/kg)+Verapamil (125mg/kg)+Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)VCR(30/ig/kg)+Verapamil (125mg/kg)+Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50 mg/kg)Survival

time(days)10.0

±0.9610.0

±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

±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

5)11.6±1.511.2±0.411.0±08.4±4.31

1.2 ±0.415.5±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

(%)100100146C168°164C163C132°148°150C144C102120C114C122°10087107115C141C138°96145C136C129C98129C116C116C10097957297134C10095134°126C122C11295121C121C117C110T/V(%)a100115a112112100112"114a109"100118112a120100107131d129"100151a142a134a100132a119a119a10076102141a108100141a133a129a118100127a127a124a116a

T/V, at each VCR dosage, the mean survival time of the treated groupdivided 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 10jug/kg was similar to that (129%) obtained in the P388/VCRbearer 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 inP388/VCR-bearing mice when VCR and verapamil were given

daily for 5 days (Table 1C). The dose of verapamil could beincreased to 125 mg/kg without manifestation of toxicity.However, VCR (200 jug/kg) with verapamil (125 mg/kg) wastoxic. 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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T. Tsuruo et al.

/ig/kg doses of VCR with verapamil (75 to 125 mg/kg). However, the effects were less than those obtained in the experiments with drug treatment for 10 successive days.

When VLB was used instead of VCR, a similar enhancementof antitumor activity of VLB occurred (Table 2). Enhancementin 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 ofthe P388/VCR bearer by approximately 30% when comparedto the group of mice treated with VLB alone (Table 2B). Although this value is less than that obtained in the experimentwith VCR, the results indicated that VCR resistance can alsobe partially overcome by VLB and verapamil in vivo.

Cellular Uptake of VCR and the Effect of Verapamil. Cellular uptake of VCR was examined in the presence of 10 nM[3H]VCR. The most prominent effect of verapamil on the cyto-

toxicity of VCR against P388/VCR cells has been obtained at10 nM VCR as is shown in Chart 1. More than 98% of P388and P388/VCR cells excluded trypan blue after treatment ofthe cells with 10 nM VCR and 6.6 juM verapamil for 5 hr.Furthermore, treatment of the cells with 6.6 fiM verapamil didnot change cellular uptake rates of a-aminoisobutyric acid and2-deoxyglucose. These results might indicate that the plasmamembrane and membrane permeability of the cells were keptintact during the drug treatment. Uptake of [3H]VCR into cul-

Table 2Effect of verapamil on antitumor activity of VLB in P388- and P388/VCR-

bearing miceEach group of 5 CD2F, mice was given i.p. implants of 106 cells of P388 or

P388/VCR leukemia on Day 0. and drugs were give i.p. daily from Days 1 to 10.

Drug anddosageA.

P388-bearingmiceControlVerapamil

(100mg/kg)VLB(30ng/kg)+Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)VLBOOfig/kg)+

Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)VLBd

Mg/kg)+Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)B.

P388/VCR-bearingmiceControlVerapamil

(100mg/kg)VLB(200,,g/kg)+Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)VLB(100/ig/kg)+

Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50mg/kg)VLB(30fig/kg)+Verapamil (100mg/kg)+Verapamil (75mg/kg)+Verapamil (50 mg/kg)Survival

time(days)10.0

±Ob8.8

±2.013.8±0.515.0±0.715.0±0.714.2±0.812.2±0.412.8

1.013.40.512.40.510.40.910.80.410.60.512.6

±3.611.8

±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±1.1T/C

(%)10088138°150°150C142C122C128°134C124C104108106126100102127C117137C136C103136C132C127°95121°115108T/V(%)a100109d109d10310010511010210010410212110092108"107"100132d128d123rf100128rf121a114"

tured P388 cells increased with time under the conditions ofconstant drug exposure (Chart 2). Approximately 0.85 pmol ofVCR was found at 5 hr in 106 P388 cells, while the amount of

VCR in P388/VCR cells was much smaller and the level almostreached a plateau (0.1 pmol/106 cells) after 1 hr of incubation;

only a marginal increase occurred thereafter. The mechanismof resistance could be explained by this phenomenon. Verapamil added to the culture at 6.6 ¿IMgreatly increased theamount of cellular VCR in both P388 and P388/VCR cells.Approximately twice the amount of VCR was found in P388cells treated with verapamil. While almost a 10-fold accumulation of VCR occurred in verapamil-treated P388/VCR cells

during 3 to 5 hr of incubation, the amount of VCR reached aslightly higher level than that in P388 cells. Enhanced cytotox-icity of VCR in P388 and P388/VCR cells by verapamil andthe overcoming of VCR resistance in P388/VCR cells in vivoand in vitro by verapamil could be explained by this phenomenon.

The enhanced accumulation of VCR in verapamil-treatedcells 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) verapamil 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 didnot show any significant effect on the binding of [3H]VCR to

tubulin, indicating that verapamil does not modify the affinity ofVCR for tubulin.

Effect of Verapamil on the Transport of VCR in P388/VCRCells. Verapamil seemed not to enhance the influx of VCR intoP388/VCR cells, inasmuch as the pretreatment of the cellswith verapamil had no effect on the cellular accumulation ofVCR (Chart 4). The efflux of intracellular VCR from P388/VCRcells, however, was significantly inhibited by verapamil as

T/V, at each VLB dosage, the mean survival time of the treated groupdivided 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

of the control experiment.Statistically significant ( p < 0.05) by Student's f test as compared with that

of mice treated with VLB alone at each dosage of VLB.

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 20mM 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

verapamil at 6.6 JIM(O O). P388/VCR cells were also incubated with VCR asabove 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]VCRincorporated into the cells were determined as described in "Materials andMethods." Cells were counted with 1-ml aliquots. Each point is the mean of

duplicate determinations.

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- i.o

0.5 1.0 1.5

Vincristine ( nmol )

2.0

Chart 3. Effect of verapamil on the binding of [3H]VCR to tubulin. Purified

tubulin (10 fig) from porcine brain was incubated in 1 ml of 0.01 M sodiumphosphate buffer, pH 6.5. containing 0.1 mM GTP, with graded concentrationsof [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 wasincubated for 15 min at 37°.The extent of binding of [3H]VCR to tubulin was thendetermined by the filter assay technique (18, 20) as described in 'Materials andMethods."

T, 2.0 -

.? 1.0 -

- 0'

Chart 4. Effect of pretreatment of P388/VCR cells with verapamil on thecellular uptake of [3H]VCR. Culture mixtures (100 ml) as described in the legendto 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 withoutverapamil. Three hr later, 10 nM [3H]VCR (specific activity, 2.8 Ci/mmol) wasadded 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 werecultivated. At time intervals, cellular uptake of I3H ]VCR was determined with

Mixture A (•)and Mixture B (O) as described in the legend to Chart 2. Eachpoint is the mean of duplicate determinations.

described below (Chart 5). The cells were preincubated with[3H]VCR and verapamil for 3 hr, and then the cells were furtherincubated at 37°or 25°with or without verapamil. At 1 hr afterincubation at 37°, about 95% of intracellular VCR was lost

from the cells incubated without verapamil; while more than70% of the drug was retained in the cells when the cells wereincubated with verapamil. At 3 and 5 hr after incubation withverapamil, approximately 45 and 30%, respectively, of theinitial amount of VCR still remained in the cells, while more than99% of intracellular VCR was lost from the cells when the cellswere incubated without verapamil. Unlabeled VCR (10 nM)added to the efflux bath had no effect on the release of

0.5 -'

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 1X 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 wascentrifuged at 80 x g for 10 min at 5°,and the precipitated cells were suspendedin 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 andD. 6.6 JIM verapamil was added. Mixtures A and B were incubated at 37°, andMixtures C and D were incubated at 25°. At time intervals and as described inthe 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 duplicatedeterminations.

intracellular VCR and its inhibition by verapamil; the velocity ofVCR release and extent of inhibition by verapamil were almostthe same, respectively, as observed above. The velocity ofefflux of intracellular VCR decreased significantly when theefflux was measured at 25°. Approximately 58, 15, and 6%,

respectively, of the initial amount of VCR remained in the cellsat 1, 3, and 5 hr after incubation at 25°. Verapamil alsoinhibited the VCR efflux at 25°. Approximately, 76, 67, and

64% respectively, of the initial amount of VCR still remained inthe cells at 1, 3, and 5 hr after incubation at 25°with verapamil.

From these results, we can state that the higher accumulationof VCR in P388/VCR cells by verapamil could occur throughan inhibition of the efflux mechanism of VCR by verapamil. Asimilar inhibition by verapamil was also observed for P388cells.

DISCUSSION

Verapamil has enhanced the cytotoxicity of VCR in bothP388 and P388/VCR cells and could completely overcomeVCR resistance in vitro. Verapamil also enhanced the chemo-therapeutic 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 wasgiven in P388/VCR-bearing mice along with verapamil, VCRresistance was almost completely overcome in vivo. Using 6.6/IM verapamil, the cellular level of VCR was enhanced to asimilar extent in both P388 and P388/VCR cells (Chart 2).Actually, in in vitro experiments, the sensitivities of P388 andP388/VCR to VCR were almost equal when 6.6 ¿IMverapamilwas added to the culture (Chart 1/4). However, in in vivoexperiments, we needed approximately 3 times the amount ofVCR to obtain a similar therapeutic effect in P388- and P388/VCR-bearing mice. A more complicated response might occurin in vivo experiments. Among the schedules of drug adminis-

MAY 1981 1971

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f. Tsuruo et al.

tration examined, the most effective therapeutic response wasobtained when VCR (or VLB) and verapamil were given togetherfor 10 or 5 successive times. Administration of VCR andverapamil on Days 1 and 5 or administration of VCR on Days1 and 5 and verapamil on Days 1,2,3, 5, 6, and 7 to P388/VCR bearers had no significant therapeutic effect. It is important that constant exposure of the resistant cells with both VCRand verapamil seems to be essential to overcome resistance.

The cellular concentrations of VCR in P388/VCR cells were3 to 5 times lower than those found in P388 cells. We observedthat the efflux of intracellular VCR occurred more rapidly forP388/VCR cells than for P388 cells. Inaba era/. (13, 14) hasreported that the mechanism of drug resistance is the activeefflux of the intracellular drug from the resistant cells. Thepossibility that verapamil alters membrane permeability couldbe denied, because verapamil did not change membrane in-tactness as determined by the trypan blue dye exclusion testand cellular uptake rates of a-aminoisobutyric acid and 2-

deoxyglucose. Furthermore, verapamil did not change the affinity of VCR for tubulin (Chart 3). We speculate that verapamilinhibits the drug efflux function of the cells and thus that a veryefficient increase in drug sensitivity could be obtained in resistant cells. The mechanisms involved in the inhibition of drugefflux by verapamil is not known, although it is presumably atemperature-dependent reaction. Oxytocin, vasopressin, insulin, adrenocorticotropin, growth hormone, and thyroid-stimu

lating hormone secretions from the cells have been suppressedby verapamil (7-9, 17, 22), although the mechanism is alsounclear 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 presumablyrelated to the mechanism of inhibition of drug efflux from thecells. For elucidation of the mechanism, we must examine theeffects of other Ca2+ antagonists using the present experimen

tal system and the effect of verapamil on the release of otheranticancer drugs and cellular components from the cells.

It might be possible to overcome the drug resistance practically by using the approach described in this paper, if activeefflux of the drug is the cause of resistance. Such a mechanismis widely observed in many experimental tumor cells (4, 6, 13,14, 23, 26, 27). The application of verapamil in practicaltherapy might be difficult as the drug possesses coronaryvasodilator activity. However, we can still speculate upon thepossibility of finding effective drugs which possess a strongerinhibitory action on drug efflux with fewer side reactions thanthose of verapamil. These possibilities might evolve from aseries of membrane-modifying agents such as Ca2+ antago

nists.

ACKNOWLEDGMENTS

We thank the Eisai Co., Ltd., and Dr. H. Sakai, University of Tokyo, for gifts ofverapamil and purified tubulin, respectively. We are indebted to H. Bowser forediting the manuscript.

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1972 CANCER RESEARCH VOL. 41

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1981;41:1967-1972. Cancer Res   Takashi Tsuruo, Harumi Iida, Shigeru Tsukagoshi, et al.   Vinblastine by Verapamil

through Enhanced Cytotoxicity of Vincristine andin Vitroand in VivoOvercoming of Vincristine Resistance in P388 Leukemia

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