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. JUNE 1983 2905 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1983 American Association for Cancer Research. T. Tsuruoetal. was incubated at 37°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 37°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. £ 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 ¡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 ¿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 ±0.2) ers.