Circumvention of Vincristine and Adriamycin Resistance in Vitro and in Vivo by Calcium Influx Blockers1

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[CANCER RESEARCH 43, 2905-2910, June 1983] 0008-5472/83/0043-0000$02.00 Circumvention of Vincristine and Adriamycin Resistance in Vitro and in Vivo by Calcium Influx Blockers1

Takashi Tsuruo,2 Harumi lida, Makiko Nojiri, Shigeru Tsukagoshi, and Yoshio Sakurai

Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Toshima-ku, Tokyo 170, Japan

ABSTRACT In various experimental tumor systems, one of the most im portant mechanisms of drug resistance, especially to Vinca al Calcium influx blockers, diltiazem, nicardipine. nifedipine, nilu- kaloid and anthracycline classes of antitumor agents, has been dipine, and nimodipine, which possess coronary vasodilator ac attributed to the enhanced drug efflux function of the resistant tivity, greatly enhanced the cytotoxicity of vincristine (VCR) in tumor cells (3, 6, 10, 16, 17, 24). These observations suggest tumor cells and especially in VCR-resistant sublines of P388 that if we could control the drug efflux function of tumor cells leukemia (P388/VCR) and human K562 myelogenous leukemia. appropriately, then we could expect anticancer agents to be The extent of enhancement was different among the drugs, and effective against resistant cells (14). We found that the cellular up to a 50- to 70-fold increase in VCR cytotoxicity occurred in calcium may be involved in the drug efflux mechanisms of the P388/VCR cells with nontoxic or marginally toxic concentrations cells (25). Some calcium influx blockers and calmodulin inhibitors of diltiazem and nicardipine. A 50- to 100-fold enhancement efficiently inhibit the VCR3 and ADM efflux function of tumor cells occurred in VCR-resistant human K562 myelogenous leukemia and especially of resistant tumor cells (24-26). VCR and ADM cells with diltiazem, nicardipine, niludipine, and nimodipine. VCR accumulate in resistant cells, and the drug resistance is circum resistance of these cell lines was circumvented completely by vented efficiently through a marked enhancement of drug cyto these blockers. Calcium influx blockers also enhanced the cyto toxicity (24-26). toxicity of Adriamycin in P388 leukemia cells and especially in its To explore the possible clinical application of this approach, Adriamycin-resistant subline. The extent of enhancement, how we have tried to determine the effectiveness of drug transport ever, was lower than that which occurred in VCR-resistant tumor modifiers from various calcium influx blockers and calmodulin lines with VCR. An approximately 10- to 30-fold increase in inhibitors. We found that calcium influx blockers are generally Adriamycin cytotoxicity occurred in P388 Adriamycin-resistant more effective than are calmodulin inhibitors. In this paper, we subline cells with diltiazem, nicardipine, niludipine, and nimodi describe the effects of several calcium influx blockers now used pine. Although VCR alone at 10 to 200 ng/kg did not confer a clinically or preclinically to circumvent VCR and ADM resistance significant therapeutic effect in P388/VCR-bearing mice, calcium in vivo and in vitro. influx blockers in doses of 30 to 125 mg/kg administered daily for 10 days with VCR enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice. A maximum of approximately MATERIALS AND METHODS

a 40 to 50% increase in life span occurred with diltiazem, Drugs. VCR and ADM, formulated for clinical use, were obtained from nicardipine, niludipine, and nimodipine. The calcium influx block Shionogi & Co., Ltd., Osaka, Japan, and Kyowa Hakko Kogyo Co., Ltd., ers also enhanced the therapeutic effect of Adriamycin in P388 Tokyo, Japan. The following calcium influx blockers were used: diltiazem Adriamycin-resistant sublime-bearing mice, although the extent (9,11), obtained from Tanabe Seiyaku Co., Ltd., Osaka, Japan; nicardi of enhancement was smaller than that observed with VCR in pine (19, 21), obtained from Yamanouchi Pharmaceutical Co., Ltd., P388/VCR-bearing mice. Tokyo, Japan; and nifedipine (4, 27), niludipine (4, 18), and nimodipine (20, 22), obtained from Bayer AG, Wuppertal-Elberfeld, Germany. INTRODUCTION Animals and Tumor Cells. Adult female BALB/c x DBA/2O F, (hereafter called CD2F,) mice weighing 20 to 23 g were obtained from In the chemotherapy of cancer of humans and animals, it has Charles River Japan, Inc., Tokyo, Japan. P388 and P388/VCR cell lines been observed widely that, although tumors respond well initially were supplied by the National Cancer Institute, NIH, Bethesda, Md. (24). The P388/ADM cell line, established at the National Cancer Institute, to certain drugs, they become progressively resistant to these NIH, was provided by Dr. Inaba (6). The human myelogenous leukemia drugs, which finally leads to therapeutic failure. Similarly, in K562 cell line (8) was provided by Dr. Ezaki, and the K562/VCR cell line experimental tumors, one of the major reasons for treatment was established in this laboratory.4 failure is the appearance and proliferation of drug-resistant tumor Cell Culture and Drug Treatment. Tumor cells were maintained in cells during the course of treatment (12, 13, 15). Because the suspension in plastic dishes (Coming Glass Works, Corning, N. Y.) in only approach to circumventing drug resistance in tumor cells at Roswell Park Memorial Institute Medium 1640 supplemented with 10% the present time is a combination therapy with other anticancer fetal bovine serum (Flow Laboratories, Stanmore, New South Wales, drugs having different modes of action, the development of a Australia) and kanamycin (100 ^g/ml) (growth medium) (24). The culture

new modality to circumvent the drug resistance in tumor cells 3The abbreviations used are: VCR, vincristine; ADM, Adriamycin; P388/VCR, could be beneficial in the chemotherapy of cancer. P388 leukemia cells resistant to vincristine; P388/ADM, P388 leukemia cells resistant to Adriamycin; K562/VCR, human myelogenous leukemia K562 cells 1This work was supported by Grants-in-akJ for Cancer Research from the resistant to vincristine; ICM. concentration of drug required for 50% inhibition of Ministry of Education, Science, and Culture (57010075) and from the Ministry of cell growth; T/C, mean survival time of the treated group divided by mean survival Health and Welfare (57-3), Japan. time of the control group. 2 Recipient of an Award from the Society for Promotion of Cancer Research, 4 T. Tsuruo, H. lida, E. Ohkochi, S. Tsukagoshi, and Y. Sakurai. Characteristics Japan. To whom requests for reprints should be addressed. and mechanisms of vincristine-resistant clones of human myelogenous leukemia Received August 17,1982; accepted March 9,1983. K562, submitted for publication.

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was incubated at 37Â°in a humidified atmosphere of 5% CO2. For the drug treatment experiments, tumor cells (2x10* for P388, P388/VCR, 100 and P388/ADM cells and 4 x 104 for K562 and K562/VCR cells) were cultured at 37Â°for 5 hr in Falcon No. 2054 culture tubes containing 2 ml of growth medium in a humidified atmosphere of 5% CO2. Then the cells 80 were treated with graded drug concentrations (0.1 to 100 HM for VCR and 10 to 104 nw for ADM) in the absence or presence of the calcium influx blockers (3.5 to 100 /IM depending on the drugs), reincubated for 60 72 hr in the presence of drugs, and counted with a Model 2BI Coulter Counter (24). Three tubes were used for each drug concentration. In the control experiment, tumor cells grew exponentially during the incubation Z 40 period, and the final cell numbers were described in each chart and table. Â£ Calcium influx blockers were dissolved in dimethyl sulfoxide at a final concentration of 100 mM and diluted with phosphate-buffered saline (0.2 8 20 M sodium phosphate:0.l5 M NaCl, pH 7.4). The final concentration of dimethyl sulfoxide in the culture was less than 0.1% (v/v), and no effect from dimethyl sulfoxide on cell growth or cell differentiation was ob o served. 100 ICso Â¡nthe presence or absence of calcium influx blockers was deter mined by plotting the logarithm of the drug concentration versus the growth rate (percentage of control) of the treated cells (23, 24). 80 Evaluation of Antitumor Activity. One-tenth ml of diluted ascites fluid containing 108 P388 and P388/VCR cells or 105 P388/ADM cells was transplanted i.p. into CD2F, mice. Calcium influx blocker and VCR or 60 ADM were dissolved in 0.9% NaCl solution [if necessary, calcium influx blockers were suspended in a small volume of 2% (w/v) methyl cellulose No. 25 (Nakarai Chemicals, Ltd., Kyoto, Japan) and diluted in 0.9% NaCl 40 solution]. The final concentration of methyl cellulose was less then 0.1%. This vehicle has no effect on the antitumor activity of VCR. Both drugs were mixed, and the mixture was administered at a constant rate of 0.02 20 ml/g body weight i.p. daily for 10 days starting from the day after tumor inoculation. Doses of calcium influx blockers, VCR, and ADM were 30 to 125 mg/kg, 10 to 400 Â¿ig/kg,and 0.5 to 1.0 mg/kg, respectively. Five O mice were used for each experimental group (24). Antitumor activity was 0.1 l 10 100 evaluated by the mean survival time of a group of mice and also Concentrationofvincristine(nM) expressed by the T/C (%) value (24). Chart 1. Effects of calcium influx blockers upon growth-inhibitory actions of VCR on P388 and P388/VCR cells. Tumor cells were treated with graded VCR RESULTS concentrations with or without calcium influx blockers 5 hr after seeding the cells at 2 x 10* per 2 ml of the medium, and cell number was counted 72 hr after the continuous drug exposure as described in "Materials and Methods." In the absence Potentiation of VCR Cytotoxicity by Calcium Influx Block of the drugs, cells grew exponentially, and the final cell numbers were (4.0 Â±0.2) ers. Calcium influx blockers showed different growth-inhibitory x 105 (S.D.) and (2.2 Â±0.1) x 10* per 2 ml of the medium for P388 and P388/ activity against various tumor cells used in this experiment. At VCR, respectively. In A. P388 cells were incubated with VCR at the indicated concentrations in the absence (â€¢)orpresence of diltiazem at 35 (A) and 100 (â€¢) the concentration studied, only niludipine at 35 MM significantly iÂ¡u,and the ICsoSof VCR were 2.10 Â±0.10, 0.60 Â±0.06, and 0.49 Â±0.02 nM, inhibited the growth of any of the cell lines derived from mice, respectively. P388/VCR cells were treated with VCR at the indicated concentrations while diltiazem and nifedipine appeared to inhibit the growth of in the absence (O) or presence of diltiazem at 35 (A) and 100 (G) JIM, and the ICMs a human-derived cell line at concentrations of 100 MM.We used of VCR were 32.4 Â±2.3, 1.12 Â±0.07, and 0.60 Â±0.19 nM, respectively. In B, P388 cells were incubated with VCR at the indicated concentrations in the absence nontoxic or slightly toxic (growth inhibition did not exceed 10%) (â€¢)orpresence of nicardipine at 3.5 (A) and 10 (â€¢)JIM,and the ICsoSof VCR were concentrations of calcium influx blockers in this experiment. 2.10 Â±0.10, 0.85 Â±0.12, and 0.27 Â±0.01 nM, respectively. P388/VCR cells were treated with VCR at the indicated concentrations in the absence (O) or presence The sensitivities of P388 and P388/VCR cells to VCR and the of nicardipine at 3.5 (A) and 10 (D) JIM, and ICsoSof VCR were 32.4 Â±2.30, 0.98 effect of dittiazem and nicardipine on these sensitivities are Â±0.12, and 0.46 Â±0.01 nM, respectively. Points mean of 3 determinations. ICsoS in the presence of each modifier for each cell line were significantly different from illustrated in Chart 1. P388/VCR cells were resistant to VCR, corresponding ICsoSin the absence of modifiers (p < 0.05 by Student's i test). and the index of resistance was approximately 15 when com pared with the ICso values of both cells (Chart 1). Diltiazem and The effect of other calcium influx blockers on the sensitivities nicardipine enhanced the cytotoxicity of VCR in P388 cells. An of P388 and P388/VCR cells to VCR was also estimated by the increase in cytotoxicity of 7.8- and 4.3-fold was produced by 10 same manner as illustrated in Chart 1. The ICso values of VCR MMnicardipine and 100 UM diltiazem, respectively, as compared in the absence or presence of calcium influx blockers were to the ICso values. These calcium influx blockers greatly poten summarized in Table 1. Niludipine and nimodipine also enhanced tiated the cytotoxicity of VCR against P388/VCR cells. Increases the cytotoxicity of VCR against P388 and especially against in VCR cytotoxicity were 70- and 54-fold at the maximum P388/VCR cells. An increase in cytotoxicity of 26-fold was concentrations of nicardipine and diltiazem, respectively. As the produced in P388/VCR cells at the maximum concentrations of ICsovalues of VCR for P388/VCR cells shifted to smaller values niludipine and nimodipine. Perfect circumvention of VCR resist than those (2.10 nw) of P388 cells by diltiazem and nicardipine, ance was also attained by these blockers. perfect circumvention of VCR resistance was attained by these These calcium influx blockers also enhanced VCR cytotoxicity blockers. against human K562 cells and especially that of VCR-resistant

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lines (Table 2). Diltiazem at 100 /Ã•Mwastoxic to human cells. A Tabte3 2- to 3-fold increase in VCR cytotoxicity occurred in K562 cells Effects of calcium influx blockers upon growth-inhibitory action of ADM on P388 and P388/ADM cells with diltiazem, nicardipine, and nimodipine as compared to the Tumor cells were treated with graded ADM concentrations with or without ICsovalues. Niludipine at 35 /Â¿Mwasmarginally toxic to human calcium influx blockers 5 hr after seeding the cells at 2 x 104 per 2 ml of the cells, differing from the effect observed for mouse cells; the most medium. Cell number was counted 72 hr after the continuous drug exposure, and the ICÂ»wasdetermined as is shown in Chart 1 (24). In the absenceof drugs, cells ^0.2) cA|juiiciiuaiiy, oiiu me MUucn nuiiiucia rcrc ^Â»*l â€”u.e/ â€¢"â€¢Ivtinu ^o o 1Effects Table respectively.Calciumx 10sper 2 ml of the medium for P388 and P388/ADM cells, of calcium influx blockers upon growth-inhibitory actions of VCR on P388 influxconcentration(MM)NoneDiltiazemocOD100Nicardipine3.510Nifedipine35100Niludipine3.510Nimodipine1035ICsoagainstP38817.3(nw)of ADM cellsTumor and P388/VCR withoutcalciumcells were treated with graded VCR concentrations with or influx blockers 5 hr after seeding the cells at 2 x 104 per 2 ml of the medium. Cell number was counted 72 hr after the continuous drug exposure, and +3.0"nn thedrugs,cells ICsowas determined as is shown in Chart 1 (24). In the absence of the Â±9753.0 grew exponentially, and the final cell numbers were (4.0 Â±0.2)x 10sand (2.2 Â±0.1)xrespectively.Calcium 105per 2 ml of the medium for P388 and P388/VCR cells, nt>yIE... â€¢* i .Â£. againstconcentration(MM)influx ICso(nw)of VCR 10.4 Â±0.767.3 Â±7.0C216

P388/VCRNone P388 +0.464.7 Â±56C31.1 Â±2.30Nifedipine35 2.10 Â±0.10* 32.4 Â±0.1"20.8 Â±1.7C260

Â±0.15Â°100 1.08 Â±0.06Â° 7.37 Â±1.617.3 Â±1.8C208 2.60Â±0.31CNiludipine3.51.14 Â±0.01" Â±1.39.0 Â±8.3Â°145

Â±0.02Â°10 1.03 + 0.02* 2.93 Â±0.5"7.3 Â±2.1C86.5 1.25Â±0.03CNimodipine100.65 Â±0.07" +0.3"10.0 Â±3.5Â°100

c35 0.98 Â±0.08* 1.63 Â±0.01 Â±0.1"5.7 +1.6e55.8 0.65 + 0.046 1.25Â±0.01Cyiew Â±0.6"P388/ADM849 Â± 3.3C " Mean Â±S.D.of 3 determinations. Mean Â±S.D.of three determinations. '' Significant (p < 0.05) by Student's t test as compared to the value without " Significant (p < 0.05) by Student's t test as compared to the value without calcium influx blocker (2.10 Â±0.10). calcium influx btocker (17.3 Â±3.0). c Significant (p < 0.05) by Student's t test as compared to the value without 0 Significant (p < 0.05) by Student's t test as compared to the value without calcium influx blocker (32.4 Â±2.30). calcium influx btocker (849 Â±97).

Table 2 prominent enhancement (5.6-fold) was observed for niludipine at Effects of calcium influx blockers upon growth-inhibitory actions of VCR on K562 35 Â»Â¿M-Thedrugs greatly enhanced the cytotoxicity of VCR and K562/VCR cells against K562/VCR cells. A more than 100-fold enhancement Tumor cells were treated with graded VCR concentrations with or without calcium influx blockers 5 hr after seeding the cells at 4 x 104 per 2 ml of the occurred with niludipine at 35 ftÂ«,and an approximately 50-fold medium. Cell number was counted 72 hr after the continuous drug exposure, and increase in cytotoxicity was observed for diltiazem, nicardipine, the ICsowas determined. In the absence of the drugs, cells grew exponentially, and the final cell numbers were (3.3 Â±0.1)x 10* and (3.2 Â±0.1)x 10* per 2 ml of and niludipine at maximum concentrations used. Perfect circum the medium for K562 and K562/VCR cells, respectively. vention of VCR resistance was attained by these blockers. Calciuminfluxconcentration againstK5622.68(nM)of VCR The effect of nifedipine was not impressive throughout these experiments. (MM)NoneDiltiazem PotentJationof ADM Cytotoxicity by Calcium Influx Block ers. Thesecalciuminfluxblockersalsoenhancedthecytotoxicity Â±0.25'1.65 Â±1.36.49 of ADM against P388 cells and especially against P388/ADM + 0.09" Â±0.57C cells. The enhancement, however, was not as prominent as that 10 observed for VCR in P388/VCR cells. An approximately 2- to 4- 35Nicardipine 1.08Â±0.19*1.57 1.14Â±0.10C2.28Â±0.18C fold increase in cytotoxicity was observed against P388 cells for diltiazem, nicardipine, niludipine, and nimodipine as compared to 3.5 Â±0.09* 10Nifedipine 1.14Â±0.03"2.49 1.08Â±0.04Â°28.2 the ICsovalues(Table 3). These calcium influx blockers enhanced prominently ADM cytotoxicity against P388/ADM cells. A 27-fold increase in cytotoxicity occurred at 10 U.Mnicardipine, and an 10 Â±0.01 Â±0.31Â° 35Niludipine 1.95Â±0.05"1.52 5.85 Â±0.07C1.17Â±0.03C approximately 10- to 15-fold increase in cytotoxicity was ob served for diltiazem, nimodipine, and niludipine. Partial circum Â±0.03" 10 vention of ADM resistance in P388/ADM cells was attained by 0.48Â±0.01*1.68Â±0.056 0.46 Â±0.06C3.79 35Nimodipine these blockers as the ICsovalues reached a range similar to that for P388 cells. Â±0.16C 10 Combined Effect of VCR and Calcium Influx Blockers on 0.98 Â±0.01*K562/VCR56.3 1.04 Â±0.01Â° 351CÂ» P388/VCR-bearing Mice. VCR administereddailyfor 10 days " Mean Â±S.D.of 3 determinations. starting from Day 1 increased the life span of P388 leukemia- 6 Significant (p < 0.05) by Student's f test as compared to the value without bearing mice. T/C values were 132, 150, 173, 223, and 238% calcium influx blocker (2.68 Â±0.25). c Significant (p < 0.05) by Student's r test as compared to the value without at VCR dosages of 10,30,100,200, and 400 /Â¿g/kg,respectively. calcium influx btocker (56.3 Â±1.3). VCR given according to the schedule above showed no ther-

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research. T. Tsuruoetal. apeutic effect in P388/vCR-bearinq mice except at dosages of overcome in the P388/VCR bearer when approximately triple 200 and 400 ug/kg, where a slightly longer survival time was amounts of VCR were given with appropriate calcium influx obtained (Charts 2 and 3). Each calcium influx blocker alone also blockers, because the T/C value of the P388/VCR bearer treated did not confer a significant therapeutic effect against P388/VCR- with VCR (100 Â¿ig/kg)and calcium influx blockers was almost bearing mice (data not shown). equal to that obtained in the P388 bearer that was treated with However, calcium influx blockers given 10 times with VCR VCR alone at 30 /Â¿g/kg. significantly increased the life span of the P388/VCR-bearing Combined Effect of ADM and Calcium Influx Blockers on mice (Charts 2 and 3). Especially notable was a 4- to 6-day P388/ADM-bearing Mice. The effect of calcium influx blockers increase in life span which was observed when diltiazem (100 to on the circumvention of ADM resistance in vivo was examined. 125 mg/kg) was administered with VCR (200 ;

Chart 2. Effect of diltiazem and nicardipine on antiiumor activity of VCR in P388/VCR- bearing mice. Each group of 5 CD2F, mice was 150 given i.p. implants of 10* cells of P388/VCR leukemia on Day 0, and drugs were given i.p. daily from Days 1 to 10 as described in "Ma _ 15 terials and Methods." A group of mice was given VCR alone at the indicated dose (â€¢);oars. S.D. In A, other groups of mice were given VCR at the indicated dose along with diltiazem at 125 (O), 100 (A), 80 (D). and 60 (O) mg/kg. 100 respectively. In B, other groups of mice were given VCR at the indicated dose along with 10 nicardipine at 75 (O), 60 (A), 45 (D). and 30 (O) mg/kg. respectively. *, significantly superior (p < 0.05 by Student's t test) to points treated with VCR alone at each dosage of VCR. Control mice survived for 11.4 Â± 1.2 days. T/C per centage value was plotted on right. 10 30 100 200 400 10 30 100 200 400 Dose of vlncrlstlne ( Â¿ig/kg )

ISO Chart 3. Effect of niludipine and nimodipine on antitumor activity of VCR in P388/VCR- bearing mice. Same experiment as in Chart 2 15 was carried out with niludipine and nimodipine. M In this experiment, control mice survived for 11.6 Â±1.1 days. Mice were given VCR alone o at the indicated dose (â€¢).bars, S.D. In A. mice were given VCR at the indicated dose along with niludipine at 75 (O), 60 (A), 45 (O), and 30 100 (O) mg/kg, respectively. In B, mice were given VCR at the indicated dose along with nimodi 10 pine at 150 (O), 125 (A), 100 (D), and 75 (O) mg/kg, respectively. â€¢,significantdifference (p < 0.05 by Student's t test) as described in Chart 2. 10 30 100 200 400 10 30 100 200 400 Dose of vlncrlstlne (

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Table 4 and in vivo which was almost comparable to that obtained by Effect of calcium influx blockers on antitumor activity of ADM in P388/ADM- verapamil (24). bearing mice Each group of 5 CD2F, mice was given i.p. implants of 105cells of P388/ADM The precise mechanism and the involvement of cellular calcium leukemia on Day 0, and drugs were given i.p. daily from Days 1 to 10. in the enhancement of VCR and ADM cytotoxicity are under investigation. Nifedipine possesses a very strong calcium influx- time dosageExperimentDrugs and (days)12.6 %100103103105â€ž117*1406127"143"138"133"127610095â€ž109"117"123"blocking action in vascular smooth muscle (5); however, the 1ControlDiltiazem effect of nifedipine in the present study was not impressive. This Â±0.5a13.0 could indicate that the calcium influx-blocking action in smooth mg/kg)Nicardipme(125 Â±013.0 muscle might be different from that in tumor cells. Actually, mg/kg)Niludipine(75 Â±013.2 mg/kg)ADM(75 Â±0.414.8 different calcium influx blockers possess different specificity for (1.0mg/kg)+ Â±0.717.6 organs or cells, and also one calcium influx blocker showed mg/kg)+Diltiazem(125 Â±1.716.0 mg/kg)+Diltiazem(100 Â±0.618.0 different response in organs and cells (5). In this study, we found mg/kg)+Nicardipine(75 Â±2.017.4 that calcium influx blockers also possessed different growth- mg/kg)+Nicardipine(60 Â±1.416.8 inhibiting activity against various tumor lines used. Diltiazem and mg/kg)+Niludipine(75 Â±1.716.0 mg/kg)ExperimentNiludipine(60 Â±013.0 nifedipine were more cytotoxic to human cells than to mouse tumor cells, while niludipine showed a stronger cytotoxicity to 2ControlNimodipine mouse tumor cells. We have not clarified the action of calcium Â±0.612.3 mg/kg)ADM(125 Â±0.514.2 influx blockers in tumor cells. However, it might be possible to (1.0mg/kg)+ Â±0.715.2+1.716.0 speculate that the calcium influx-blocking action of the drugs is mg/kg)+Nimodipine(100 Nimodipine(75 mg/kg)Survival Â±0.6T/C involved in the enhancement of the antitumor activity in tumor 8 Mean Â±S.D. cells, since all calcium influx blockers examined possessed an " Statistically significant (p < 0.05) by Student's i test as compared with that of impressive effect. We have speculated that the efflux of VCR the control experiments. and ADM from tumor cells, especially from drug-resistant cells, is possibly controlled by the calcium-calmodulin complex (25); in life span by calcium influx blockers was approximately 3 days. however, the real mechanism remains to be discerned. We are Similar enhancement of chemotherapeutic effect also occurred presently investigating more closely the action of cellular calcium with ADM at 0.5 mg/kg along with the calcium influx blockers in the drug transport mechanisms of tumor cells in order to (data not shown). Although this value is less than that obtained determine the validity of our speculation. in the experiment with VCR in P388/VCR bearers, these results The present approach using calcium influx blockers in cancer indicate that calcium influx blockers can render the ADM-resist- chemotherapy has at least the 4 following advantages: (a) cir ant tumor cells partly susceptible to ADM in vivo. cumvention of drug resistance in tumor cells; (b) circumvention of heterogeneity of tumor cells in drug response; (c) effectiveness against various antitumor agents which are transported outside DISCUSSION the cells by the same efflux mechanism; and (d) dose deescala We have reported previously that verapamil, a calcium influx tion of antitumor agents. We are currently undertaking a preclin- blocker, enhanced the cytotoxicity of VCR and vinblastine in ical toxicology study of these calcium influx blockers, including P388 and P388/VCR cells and that VCR resistance of P388/ verapamil, to explore the possibility of applying the present VCR cells could be overcome in vivo and In vitro (24). In a variety approach clinically for the circumvention of drug resistance. of tumor cells, Vinca alkaloid-resistant cells are also resistant to anthracyclines (1-3,7,17,28). Both classes of antitumor agents ACKNOWLEDGMENTS are actively transported outside the tumor cells and especially We thank Tanabe Seiyaku, Co., Ltd., Yamanouchi Pharmaceutical Co., Ltd., those that are drug resistant (3, 6,10,16, 17, 24). Subsequent Bayer AG, Germany, for their gifts of calcium influx blockers. We are indebted to studies revealed that verapamil enhances the cytotoxicity of VCR E. Sass for editing and to M. Shimizu for typing the manuscript. in P388/ADM cells and also enhances the cytotoxicity of ADM in P388/VCR and P388/ADM cells in vitro (26). Calmodulin REFERENCES inhibitors also effectively enhance VCR and ADM cytotoxicity in 1. Siedler, J. L. and Riehm. H. Cellular resistance to actinomydn D in Chinese P388/VCR and P388/ADM (25). We found that verapamil and hamster cells In vitro: cross-resistance, radtoautographic, and cytogenetic calmodulin inhibitors efficiently inhibit the VCR- and ADM efflux studies. Cancer Res., 30. 1174-1184,1970. 2. Dane, K. Cross-resistance between Vinca alkaloids and anthracyclines in function of tumor cells and especially of resistant cells (25). VCR Ehrlich asertes tumor in vivo. Cancer Chemother. Rep. Part I, 56: 701-708, and ADM accumulated greatly in resistant tumor cells and re 1972. 3. Dane, K. Active outward transport of daunomycin in resistant Ehrlich ascites sulted in the marked enhancement of drug cytotoxicity in drug- tumor cells. Btochim. Biophys. Acta, 323: 464-483,1973. resistant tumor cells (24-26). 4. Fleckenstein,A., Fleckenstein-GÃ¼n,G.,Byon, Y. K., Haastert, H. P., and SpÃ¤h, F. VergleichendeUntersuchungen Ã¼berdieCa**-antagonistischen Grundwir These results suggest the possibility of using this approach to kungen von Niludipin (Bay a 7168) und Nifedipin (Bay a 1040) auf Myokard, overcome the drug resistance clinically. In order to find out which Myomertrium und glatte Gefassmuskulatur. Arzneim. Forsch., 29: 230-246, drugs are potentially effective clinically, we examined various 1979. 5. GrÃ¼n,G.,and Fleckenstein, A. Die etektromechanische Entkoppelung der calcium influx blockers and calmodulin inhibitors. Among those glatten Gefassmuskulatur als Grundprinzip der Koronardilatation durch 4-(2- drugs examined, calcium influx blockers generally were more Nitrophenyl)-2,6-dirnethyl-1,4-dihydropyridine-3,5-dicarbonsaure-dirnethyles- effective than calmodulin inhibitors, especially in in vivo experi ter (BAY a 1040, Nifedipine).Arzneim. Forsch., 22: 334-342,1972. 6. Inaba, M., Kobayashi, H., Sakurai, Y., and Johnson, R. K. Active efflux of ments. Among calcium influx blockers, diltiazem, nicardipine, daunomycin and Adriamycin in sensitive and resistant subÃ®mesofP388 leu niludipine, and nimodipine showed an impressive effect in vitro kemia. Cancer Res., 39:2200-2203,1979.

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7. Johnson, R. K., Chitnis. M. P., Embrey. W. M., and Gregory, E. B. Character 19. Takenaka. T., Usuda, S . Nomura, T., Maeno, H., and Sado, T. Vasodilator istics m vivo of resistance and cross-resistance of an adnamycm-resistant profile Of a new 1,4-dihydropyndine derivative, 2.6-d;methyl-4-(3-mtrophenyl)- sublmeof P388 leukemia. Cancer Treat. Rep., 62:1535-1547,1978. 1,4-dihydropyndine-3,5-dicarboxylic acid 3-12-

2910 CANCER RESEARCH VOL. 43

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research. Circumvention of Vincristine and Adriamycin Resistance in Vitro and in Vivo by Calcium Influx Blockers

Takashi Tsuruo, Harumi Iida, Makiko Nojiri, et al.

Cancer Res 1983;43:2905-2910.

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