Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
[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 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1981 American Association for Cancer Research. 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 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1981 American Association for Cancer Research. 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- MAY 1981 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1981 American Association for Cancer Research. 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 CANCER RESEARCH VOL. 41 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1981 American Association for Cancer Research. 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 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1981 American Association for Cancer Research. 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 tumor cells. Biochim. Biophys. Acta, 323. 466-483, 1973. 7. Devis, G., Somers, G., Van Obberghen, E., and Malaisse, W. J. Calcium antagonists and islet function. I. Inhibition of insulin release by verapamil. Diabetes, 24: 547-551, 1975. 8. Dreifuss, J. J., Grau. J. D., and Nordmann, J. J. Calcium movements related to neurohypophysical hormone secretion. In: E. Carafoli (ed.). Calcium Transport in Contraction and Secretion, pp. 271-279. Amsterdam: NorthHolland Publishing Co., 1975. 9. Eto, S., Wood. J. M., Hutchins. M., and Fleischer, N. Pituitary "5Ca* * uptake 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. nists. 24. 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. 26. REFERENCES 27. 1. Allison, A. C. The role of microfilaments and microtubules in cell movement, endocytosis and exocytosis. In: M. Abercrombie (ed.), Locomotion of Tissue Cells. CIBA Foundation Symposium, Vol. 14, p. 109. New York: Associated Scientific Publishers, 1973. 1972 28. and release of ACTH, GH, and TSH: effect of verapamil. Am. J. Physiol.. 226. 1315-1320, 1974. Fleckenstein, A. Specific inhibitors and promotors of calcium action in the excitation-contraction coupling of heart muscle and their role in the preven tion or production of myocardial lesions. In: P. Harris and L. Opie (eds.), Calcium and the Heart, pp. 135-138. New York: Academic Press Inc., 1971. Fleckenstein, A. Specific pharmacology of calcium in myocardium, cardiac pacemakers, and vascular smooth muscle. Annu. Rev. Pharmacol. Toxicol.. 17: 149-166, 1977. Goldman, R. D., and Knipe, D. M. Function of cytoplasmic fibers in nonmuscle cells. Cold Spring Harbor Symp. Quant. Biol., 37: 523-534, 1972. Inaba, M., Kobayashi, H., Sakurai, Y., and Johnson, R. K. Active efflux of daunomycin and Adriamycin in sensitive and resistant sublines of P388 leukemia. Cancer Res., 39: 2200-2203. 1979. Inaba, M., and Sakurai, Y. Enhanced efflux of actinomycin D, vincristine, and vinblastine in Adriamycin-resistant subline of P388 leukemia. Cancer Lett., 8: 111-115, 1979. Kohlhardt, M., Bauer, P., Krause, H., and Fleckenstein, A. Differentiation of the transmembrane Na and Ca channel in mammalian cardiac fibers by the use of specific inhibitors. Pfluegers Arch. Gesamte Physiol. Menschen Tiere, 335: 309-322, 1972. Langer, G. A., Serena. S. D., and Nudd, L. M. Localization of contractiledependent Ca: comparison of Mn and verapamil in cardiac and skeletal muscle. Am. J. Physiol.. 229: 1103-1107, 1975. Matthews, E. K., and Sakamoto, Y. Electrical characteristics of pancreatic islet cells. J. Physiol., 246: 421-437, 1975. Owellen, R. J., Donigian, D. W., Hartke, C. A., Dickerson, R. M., and Kuhar, M. J. Binding of vinblastine to tubulin and to particulate fractions of mam malian brains. Cancer Res., 34: 3180-3186. 1974. Owellen, R. J., Hartke, C. A., Dickerson, R. M., and Hains, F. O. Inhibition of tubulin-microtubule polymerization by drugs of the Vinca alkaloid class. Cancer Res., 36: 1499-1502. 1976. Owellen, R. J., Owens, A. H., Jr., and Donigian, D. W. The binding of vincristine, vinblastine and colchicine to tubulin. Biochem. Biophys. Res. Commun., 47. 685-691, 1972. Porter, K. R. Cytoplasmic microtubules and their functions. In: G. E. Wolstenholme and M. O'Connor (eds.), Principles of Biomolecular Organization, pp. 308-356. Boston: Little, Brown & Co., 1966. Rüssel,J. T., and Thorn, N. A. Calcium and stimulus-secretion coupling in the neurohypophysis. II. Effect of lanthanum, a verapamil analog (D600) and prenylamine on 45-calcium transport and vasopressin release in isolated rat neurohypophyses. Acta Endocrinol., 76: 471-485. 1974. See, Y. P., Carlsen, S. A., Till, J. E., and Ling. V. Increased drug permeability in Chinese hamster ovary cells in the presence of cyanide. Biochim. Biophys. Acta, 373: 242-252, 1974. Shelanski, M. L., Gaskin. F., and Cantor, C. R. Microtubule assembly in the absence of added nucleotides. Proc. Nati. Acad. Sei. U. S. A., 70: 765768, 1973. Sieber, S. M.. Mead, J. A. R., and Adamson, R. H. Pharmacology of antitumor agents from higher plants. Cancer Treat. Rep.. 60: 1127-1139 1976. Skovsgaard, T. Transport and binding of daunorubicin, Adriamycin, and rubidazone in Ehrlich ascites tumor cells. Biochem. Pharmacol., 26: 215222, 1977. Skovsgaard, T. Mechanism of resistance to daunorubicin in Ehrlich ascites tumor cells. Cancer Res., 38: 1785-1791, 1978. Tsuruo, T., lida, H., Tsukagoshi, S., and Sakurai, Y. Comparison of cytotoxic effect and cellular uptake of 1-/8-o-arabinofuranosylcytosine and its W-acyl derivatives, using cultured KB cells. Cancer Res., 39: 1068-1070, 1979. CANCER RESEARCH VOL. 41 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1981 American Association for Cancer Research. 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. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/41/5/1967 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1981 American Association for Cancer Research.