Effects of Quinidine and Related Compounds on Cytotoxicity and Cellular Accumulation of Vincristine and Adriamycin in Drug- Resistant Tumor Cells1

[CANCER RESEARCH 44, 4303-4307, October 1984]

Effects of Quinidine and Related Compounds on Cytotoxicity and Cellular Accumulation of Vincristine and Adriamycin in Drug- resistant Tumor Cells1

Takashi Tsuruo,2 Harumi lida, Yoshiko Kitatani, Keiko Yokota, Shigeru Tsukagoshi, and Yoshio Sakurai

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

ABSTRACT characteristics of tumor cells. We have reported that calcium channel blockers and calmod- Quinidine, which has antiarrhythmic activity, greatly enhanced ulin inhibitors increase cellular accumulation of VCR3 and ADM the cytotoxicity of vincristine (VCR) in tumor cells and especially in induced drug-resistant or inherently drug-resistant tumor cells in VCR-resistant sublines of P388 leukemia (P388/VCR) and (24,25,29-32) by inhibiting outward transport of these antitumor human myelogenous leukemia. A nontoxic concentration of quin- agents (24, 25, 29, 30), thereby causing reversal of drug resist idine increased VCR cytotoxicity in these resistant tumor cells ance in the tumor cells (24-27, 29-32). These observations, about 50 to 80 times, and the drug in combination with VCR together with previous findings, suggest that the drug efflux could completely reverse VCR resistance of these cell lines. mechanism in the plasma membrane of the resistant tumor cells Quinidine also enhanced the cytotoxicity of Adriamycin, espe cially in the Adriamycin-resistant subline of P388 leukemia; this might be related to the function of cellular calcium, as well as to enhancement (8-fold) was less than that of VCR toxicity in the changes in the plasma membrane (4, 14, 19). In further studies VCR-resistant tumor line. When administered daily for 10 days on this mechanism, we examined the effects on cellular accu mulation of VCR and ADM of various drugs that interact with with VCR, quinidine at doses of 50 to 125 mg/kg significantly enhanced the chemotherapeutic effect of VCR in P388/VCR- the membrane but do not block calcium channels. We found that antiarrhythmic agents, especially quinidine, have potent effects bearing mice. Some other antiarrhythmic agents also showed on the cytotoxicities and cellular accumulations of VCR and similar effects in vitro, but these effects were considerably lower ADM. than that of quinidine. Quinidine increased the cellular levels of VCR and daunomycin in VCR-resistant sublines of mouse and human tumors and the MATERIALS AND METHODS ADM-resistant mouse tumor line in vitro, respectively. Quinidine Drugs. VCR and ADM for clinical use were obtained from Shionogi also enhanced the cellular accumulation of VCR in P388/VCR and Co., Osaka, Japan, and Kyowa Hakko Kogyo Co., Tokyo, Japan, cells in vivo. Thus, the therapeutic effect observed in P388/VCR- respectively. [G-3H]VCR sulfate (5.8 Ci/mmol) and [G-3H]DAU (4.34 Ci/ bearing mice might be due to the enhanced accumulation of mmol) were purchased from the Radiochemical Center, Amersham, VCR in P388/VCR cells by quinidine. The increase of cellular England. Quinidine sulfate, propranolol hydrochloride, 5,5-diphenylhy- accumulation of VCR was partly explained by inhibition of efflux dantoin sodium salt, lidocaine, and ajimalin were purchased from Nakarai of VCR and daunomycin from the resistant tumor cells. The Chemical Co., Kyoto, Japan. Procainamide was a product of Sigma mechanism of this phenomenon is discussed in relation to pre Chemical Co., St. Louis, MO. Animals and Tumor Cells. Adult female BALB/c x DBA/2Cr F, vious findings on calcium channel blockers. (hereafter called CD2F,) mice weighing 20 to 23 g were obtained from Charles River Japan, Inc., Tokyo, Japan. P388, P388/VCR, and P388/ INTRODUCTION ADM cells lines were supplied by the National Cancer Institute, NIH, Bethesda, MD (26). The human myelogenous leukemia K562 cell line In experimental systems, it is known that anthracycline-resist- (17) was provided by Dr. Ezaki, and the K562/VCR cell line was estab ant tumor cells are also resistant to other DNA intercalating lished in this laboratory (28). drugs and to mitotic spindle poisons, including Vinca alkaloids, Cell Culture and Drug Treatment. Tumor cells were maintained in inhibitors of protein synthesis, and other structurally unrelated suspension in plastic dishes (Coming Glass Works, Coming, NY) in compounds (3, 5-7, 13, 16, 21, 33). The molecular mechanism Roswell Park Memorial Institute Medium 1640 supplemented with 10% of this pleiotropic phenotype of drug resistance remains far from fetal bovine serum (Flow Laboratories, Stanmore, New South Wales, Australia) and kanamycin (100 /Â¿g/ml)(growth medium) (26,29). Cultures clear; however, these pleiotropic drug-resistant tumor cells pos were incubated at 37Â°in a humidified atmosphere of 5% CO2 in air. For sess an enhanced drug efflux function common to various anti- experiments on the effects of drugs, tumor cells (P388, 2 x 104; P388/ tumor agents described above (7, 12, 18, 20, 21, 29). Recent VCR, 3 x 104; P388/ADM, 2 x 104; and K562 and K562/VCR, 4 x 104) findings (4, 14, 19) have suggested that the mechanism of were cultured at 37Â°for 5 hr in Falcon No. 2054 culture tubes containing pleiotropic drug resistance is closely related to the expression of 2 ml of growth medium in a humidified atmosphere of 5% CO2 in air. a glycoprotein with a molecular weight of about 170,000 on the Then, the cells were treated with graded concentrations of VCR (0.1 to cell surface. Development of pleiotropic drug resistance of tumor 100 nw) or ADM (0.01 to 10 /Â¿M)inthe absence or presence of antiar cells is now believed to be related to changes in membrane rhythmic drugs (1.0 to 100 MM,depending on the drug), reincubated for 72 hr in the presence of drugs, and counted in a Model ZBI Coulter 1This work was supported by grants-in-akJ for cancer research from the Ministry of Education, Science, and Culture (57010075) and from the Ministry of Health and 3The abbreviations used are: VCR, vincristine; ADM, Adriamycin; DAU,dauno Welfare (57-3), Japan. mycin; P388/VCR, P388 leukemia cells resistant to vincristine; P388/ADM, P388 2 Recipient of an award from the Society for Promotion of Cancer Research, leukemiacells resistant to Adriamycin; K562/VCR, human myelogenous leukemia Japan. To whom requests for reprints should be addressed. K562 cells resistant to vincristine; 1CÂ»,concentrationof drug required for 50% Received September 19, 1983; accepted June 25,1984. inhibitionof cell growth.

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Counter (29). Three tubes were used for each drug concentration. In the of 10 fiM, it inhibited the growth of mouse cell lines only slightly control experiment, tumor cells grew exponentially during the incubation (<5%) and had no effect on growth of human-derived cell lines, period; their final cell numbers are given in each chart and table. Drugs while at a concentration of 30 /UM,it caused about 10% growth were dissolved in dimethyl sulfoxide at a final concentration of 100 ITIM and diluted with phosphate-buffered saline (0.2 M sodium phosphate:0.15 inhibition. The sensitivities of P388 and P388/VCR cells to VCR and the M NaCI, pH 7.4). The final concentration of dimethyl sulfoxide in the culture was less than 0.1% (v/v), which had no detectable effect on cell effect of quinidine on these sensitivities are illustrated in Chart growth. 1. P388/VCR cells were resistant to VCR, and the index of The ICsoSin the presence or absence of drugs were determined by resistance was approximately 16, as determined by comparison plotting the logarithm of the drug concentration versus the growth rate of the ICsovalues for the 2 cell lines (Chart 1). Quinidine enhanced (percentage of control) of the treated cells (26, 29). the cytotoxicity of VCR in P388 cells; at 10 MM,it increased the Evaluation of Antitumor Activity. A sample of 0.1 ml of diluted ascites cytotoxicity 2-fold as compared with the IC50value. It also greatly fluid containing 106 P388/VCR cells was transplanted i.p. into CD2F, increased the cytotoxicity of VCR against P388/VCR cells, the mice. VCR was dissolved in 0.9% NaCI solution. Quinidine was sus increase being 82-fold at 10 UM quinidine. As quinidine shifted pended in a small volume of 2% (w/v) methyl cellulose (No. 25; Nakarai the IC50value of VCR for P388/VCR cells to smaller values than Chemicals, Kyoto, Japan) and diluted with 0.9% NaCI solution. The final that (1.78 nM) of P388 cells, it completely overcame the VCR concentration of methyl cellulose was less than 0.1%. This vehicle had no effect on the antitumor activity of VCR. The 2 drugs were mixed, and resistance of these cells. the mixture was administered at a dose of 0.02 ml/g body weight i.p. The effects of quinidine on the sensitivities of K562 and K562/ daily for 10 days starting from the day after tumor inoculation (26, 29). VCR cells to VCR and of P388 and P388/ADM cells to ADM The doses of quinidine and VCR were 50 to 125 mg/kg and 100 to 200 were also examined in the same manner, as illustrated in Chart /ig/kg. respectively. Groups of 10 mice were used in the experiments. 1. The ICsovalues of VCR and ADM in absence and presence of As described previously (26, 29), quinidine are summarized in Table 1. Quinidine enhanced VCR Mean survival time of treated mice cytotoxicity against human K562 cells and especially VCR-re- Antitumor activity (%) = x 100. Mean survival time of control mice sistant lines (Table 1). It caused a 2-fold increase in VCR cyto Cellular Uptake of [3H]VCR and [3H]DAU in the Presence of Quini toxicity in K562 cells as determined from ICso values. It greatly enhanced the cytotoxicity of VCR against K562/VCR cells, caus dine. Cellular uptake of the drugs was measured in growth medium (30). ing 50-fold enhancement at 10 /IM. Thus, it could completely The effects of quinidine were compared at the same nontoxic concentra tion; tumor cells (2 x 10s) in Falcon No. 2054 culture tubes containing 1 overcome the VCR resistance of these cells. Quinidine also ml of growth medium with 20 ITIM 4-(2-hydroxyethyl)-1-piperazineeth- enhanced the cytotoxicity of ADM, especially on P388/ADM cells, anesulfonic acid buffer were incubated at 37Â°in the presence of quinidine causing an 8-fold increase in ADM cytotoxicity at 10 Â¿IM;how with [3H]VCR (10 nw; specific activity, 5.8 Ci/mmol) and [3H]DAU (10 nn/i; ever, it had a negligible effect in P388 cells. Quinidine only specific activity, 4.34 Ci/mmol), respectively, and the amounts of intra- partially reversed ADM resistance in P388/ADM cells, because cellular VCR and DAU were measured at various times as described the IC50value did not reach a similar range to that for P388 cells. previously (29, 30). Cellular uptake of VCR in the presence of quinidine was also deter mined in animal experiments. In this case, experimental conditions were similar to those of therapeutic experiments. Groups of mice (3 mice/ group) were inoculated i.p. with 106 P388 or P388/VCR cells. One week later, when both cell lines (P388 and P388/VCR) were in good condition and had reached approximately 1.2 to 1.6 x 10* cells/mouse, mice were given i.p. injections of [3H]VCR (0.2 mg/kg; specific activity, 2.0 Ci/mmol) and quinidine (100 mg/kg) as described in the therapeutic experiments. After 30 min, tumor cells were recovered from the peritoneal cavity, and the intracellular VCR was determined as mentioned above. Efflux of [3H]VCR or [3H]DAU and Effect of Quinidine. VCR- and ADM-resistant tumor cells carry out efflux of VCR and DAU to the outside of the cell and usually maintain very low intracellular levels of these drugs (7,12, 18, 20, 21, 29). To obtain sufficiently high intracellular concentra tions of drugs, we initially loaded P388/VCR, K562/VCR, and P388/ ADM cells with 33 nw [3H]VCR and 33 nM [3H]DAU in glucose-free 100 growth medium supplemented with 10% dialyzed fetal bovine serum Concentration of vincristine (nM) (Grand Island Biological Co.) in the presence of 10 mM sodium azide for Chart 1. Effects of quinidine on the growth-inhibitory actions of VCR on P388 30 min as described previously (12, 20, 30). Then, each cell suspension and P388/VCR cells. Tumor cells were treated with graded VCR concentrations was centrifuged (80 x g, 10 min, 5Â°),and the precipitated cells (2 x 10s) with or without quinidine 5 hr after seeding the cells at 2 x 104 (P388) and 3 x 104 (P388/VCR) per 2 ml of medium, and cell numbers were counted after 72 hr of were resuspended (>98% of the cells excluded trypan blue) in 1 ml of continuous exposure to drugs as described in "Materials and Methods." In the growth medium containing 20 ITIM 4-(2-hydroxyethyl)-1-piperazineetha- absence of drugs, the cells grew exponentially, and the final cell numbers of P388 nesulfonic acid buffer and incubated at 37Â°in the absence or presence and P388/VCR were (2.75 Â±0.03) (S.D.) x 105 and (2.35 Â±0.04) x 105 per 2 ml of quinidine. At the indicated times, the intracellular [3H]VCR and [3H]- of medium, respectively. When P388 cells were incubated with VCR at the indicated DAU levels were determined as described previously (29, 30). concentrations in the absence (â€¢)orpresence of quinidine at 1 (A), 3 (â€¢).or10 (Â») >iM,the ICsoSof VCR were 1.78 Â±0.09,1.27 Â±0.04, 0.99 Â±0.02, and 0.85 Â±0.01 nM, respectively. When P388/VCR cells were treated with VCR at the indicated RESULTS concentrations in the absence (O) or presence of quinidine at 1 (A), 3 (D), and 10 (O) litÂ»,the ICsoSof VCR were 28.8 Â±1.47, 22.2 Â±3.2,1.49 Â±0.28, and 0.35 Â± Potentjation of VCR and ADM Cytotoxicity by Quinidine. 0.01 nw, respectively. Points, mean for 3 determinations. All IC^s in the presence of quinidine, except that for 1 UM on P388/VCR, were significantly different from The various tumor cells used in these experiments demonstrated the corresponding ICsoSwithout quinidine (p < 0.05 by Student's f test). Quinidine equivalent cytotoxic responses to quinidine. At a concentration alone at 1 to 10 ^M was nontoxic.

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Table 1 Enhancement of cytotoxicities of VCR and ADM by quinidine in drug-sensitive and -resistant tumor cells Tumor cells were treated with graded concentrations of VCR or ADM with or without quinidine 5 hr after seeding the cells at 4 x 104 (K562 and K562/VCR cells) or 2 x 104 (P388 and P388/ADM cells) per 2 ml of medium. Cell numbers were counted after 72 hr of continuous drug exposure, and the ICM was determined as shown in Chart 1 (25, 28). In the absence of drugs, cells grew exponentially, and the final cell numbers of K562, K562/VCR, P388, and P388/ADM were (1.98 Â± 0.03) x 105, (1.75 Â±0.04) x 105, (2.91 Â±0.03) x 105, and (3.02 Â±0.08) x 105 per 2 ml of medium, respectively. ICÂ»(nM) of VCR against ICÂ»(nw) of ADM against Quinidine (,"")013 Time ( hr ) 72 Â±0.23s Â±5 Â±1.15 Â±80.0 Chart 2. Effects of quinidine on the cellular accumulation of [3H]VCR and [3H]- 16.9 Â±1.64" 204 Â±34.06 1.56 Â±0.08 14.3 Â±1.46 DAU. P388/VCR (A). K562/VCR (B), or P388/ADM (C) cells (each 2 x KP/ml 3.89 Â±0.10* 119 Â±12.7* 1.08 Â±0.19 15.7 Â±0.78 medium) were incubated in Roswell Park Memorial Institute Medium 1640 contain 10K5621.1.01 Â±0.24K562/VCR1102.26 Â±0.07*P38817.314.4 Â±1.96P388/ADM71389.1 Â±16.3* ing 10% fetal bovine serum and 20 mu 4-(2-hydroxyethyl)-1-piperazineethanesul- a Mean Â±S.D. of 3 determinations. fonic acid buffer at 37Â°with 10 nM [3H]VCR (specific activity, 5.8 Ci/mmol) in A and b Significant difference (p < 0.05) by Tukey's test from the value without B or [3H]DAU (specific activity, 4.34 Ci/mmol) in C in the absence (â€¢)orpresence of quinidine at 3 JIM(O) or 10 JIM(A). At the indicated times, the intracellular [3H]- quinidine in each cell line. VCR or [3H]DAU levels were determined as described previously (28, 29). Points, mean for triplicate determinations. The significance of differences in points from Table 2 the corresponding values without quinidine (*, P < 0.05) was analyzed by the ( Effect of quinidine on antitumor activity of VCR in P388/VCR-bearing mice test. Groups of 10 CD2F, mice were given i.p. implants of 10" cells of P388/VCR leukemia on Day 0, and drugs were given i.p. daily from Day 1 to Day 10. Table 3 dosageControlQuinidineDrug and time(days)10.3 (%)100102119 Effect of quinidine on the intracellular accumulation of pHJVCR in mice Groups of mice (3 mice/group) were inoculated with 10e P388 or P388/VCR Â±0.5*10.5 cells. One week later, mice were given injections i.p. of [3H]VCR (0.2 mg/kg; specific activity, 2 Ci/mmol) with or without quinidine (100 mg/kg). After 30 min, mg/kg)VCR(125 Â±1.012.3 tumor cells were recovered from the peritoneal cavity. The amount of intracellular (200Mg/kg)+ Â±1.214.5Â±1.1C VCR was determined as described previously (29, 30). Quinidine(125 mg/kg) 141 14.4 Â±1.6Â° VCR + Quinidine(100 mg/kg) 140 TumorcellP388 administration+Intracellular (pmol/10'cell)3.27 + Quinidine(75 mg/kg) 13.9 Â±1.0 135 Â±0.30" mg/kg)VCR(100^g/kg)+ Quinidine(50 13.6Â±0.711.0 132107 P388/VCR 1.12 Â±0.05 Â±1.2 P388/VCRQuinidine 2.94 Â±0.06 13.5Â±1.0C + Quinidine(125 mg/kg) 131 Mean Â±S.D. of the values obtained from 3 mice. + Quinidine(100 mg/kg) 13.0Â±0.9C 126 13.1 Â±0.9C + Quinidine(75 mg/kg) 127128 13.2 Â±0.4CT/Ca + Quinidine(50 mg/kg)Survival 2 hr resulted in about 4- to 5-fold accumulation of VCR in P388/ 8 T/C, antitumor activity of treated versus control mice. 6 Mean Â±S.D. VCR cells (Chart 2A). The effects of quinidine in enhancing the c Significant difference (p < 0.05) by Tukey's test from the respective value cytotoxicity of VCR in P388/VCR cells and overcoming the obtained with VCR alone at 200 or 100 fig/kg. resistance in P388/VCR cells to VCR in vivo and in vitro could be explained by this phenomenon. Combined Effect of VCR and Quinidine on P388/VCR- Similar enhancement of the cellular uptake of VCR by quinidine bearing Mice. VCR administered daily for 10 days starting from was also observed in an animal experiment. In this experiment, Day 1 increased the life span of P388 leukemia-bearing mice. the doses of quinidine and VCR were 100 and 0.2 mg/kg, The antitumor activity of treated versus control mice was 173 respectively, to mimic the therapeutic experiment. After 30 min and 223% at VCR dosages of 100 and 200 /*g/kg, respectively. of [3H]VCR administration, an average of 1.12 pmol of VCR was VCR given according to the above schedule had little effect on found in P388/VCR cells, while 3.27 pmol of VCR was found in P388/VCR-bearing mice, and at 200 /Â¿g/kg,Â¡tcaused slight P388 cells (Table 3). The intracellular VCR level is clearly reduced increase in the survival time (Table 2). Quinidine alone also did in resistant cells as has been widely observed in in vitro experi not have a significant therapeutic effect on P388/VCR-bearing ments (6, 21, 29, 33). These values are higher than those mice. However, quinidine given 10 times with VCR significantly observed in in vitro experiments (Chart 2A), because the VCR increased the life span of P388/VCR-bearing mice (Table 2). The concentration in the peritoneal cavity in mice (VCR was given at result was not impressive, but an increase in life span of 2 to 3 0.2 mg/kg) might be higher than that of the cellular experiment days was observed in mice treated with quinidine and VCR. (10 nM). When quinidine was given to the P388/VCR-bearing Effects of Quinidine on Cellular Accumulation of [3H]VCR mice at 100 mg/kg with VCR at 0.2 mg/kg, 2.94 pmol of [3H]- and [3H]DAU in Vitro and in Vivo. Cellular uptake of VCR was VCR was found in 106 P388/VCR cells. Thus, approximately 2.6- examined in the presence of 10 nM [3H]VCR or [3H]DAU. More fold accumulation of [3H]VCR occurred in P388/VCR cells. This than 98% of the P388 and P388/VCR cells excluded trypan blue enhancement was almost similar to that observed in in vitro after treatment with 10 nM antitumor drug and 10 /Â¿Mquinidine experiments (Chart 2). These results indicate that similar reac for 2 hr. Uptake of [3H]VCR into cultured P388/VCR cells almost tions could occur in both in vitro and in vivo experiments by reached a plateau after 30 min under the condition of constant quinidine. Potentiation of the VCR effect in vivo by quinidine drug exposure (Chart 2). Approximately 0.1 pmol of VCR was could also be explained by the quinidine-induced accumulation found at 1 hr in 106 P388/VCR cells. Quinidine added to the of VCR in resistant tumor cells. culture at 3 or 10 U.Mgreatly increased the amount of cellular Quinidine enhanced cellular uptake of [3H]VCR similarly in VCR in P388/VCR cells. Treatment with 10 MMquinidine for 1 to K562/VCR cells (Chart 2B). The cellular level of VCR in K562/

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VCR cells was less than that in P388/VCR cells, and in fact, VCR at 1 hr after the incubation under the conditions described VCR resistance of K562/VCR was stronger than that of P388/ in Chart 2 (data not shown). These values were considerably VCR cells (Chart 1; Table 1). However, more than 10-fold accu higher than those observed in resistant P388 and K562 cells. mulation of VCR occurred in 1 to 2 hr during treatment of K562/ Quinidine at 10 MMenhanced VCR and DAU accumulation 1.5- VCR cells with 10 MMquinidine. and 1.2-fold in P388 cells, respectively (data not shown). The Quinidine enhanced the uptake of [3H]DAU by P388/ADM cells drug also enhanced VCR accumulation 1.3-fold in K562 cells. (Chart 2C). In this experiment, we used [3H]DAU instead of [3H]- This enhancement, however, was considerably smaller than that ADM, because the latter compound is not available commercially, observed for resistant tumor cells (Chart 2). and P388/ADM cells show almost equal resistance to DAD and Effect of Quinidine on Efflux of VCR and DAU in Resistant ADM. Uptake of [3H]DAU into P388/ADM cells almost reached a Tumor Cells. [3H]VCR and [3H]DAU accumulated in resistant plateau after 10 min of incubation, and only a very slight increase tumor cells in the presence of sodium azide in glucose-free occurred thereafter. Quinidine at 3 or 10 /tw greatly enhanced growth medium (30) and were rapidly reduced on incubation of the cellular accumulation of DAU in resistant cells. These con the cells in fresh medium (Chart 3). On incubation for 1 hr, about centrations of quinidine caused about 5- to 6-fold accumulation 80 to 90% of the intracellular VCR was lost from P388/VCR and of VCR. The effect of 3 fiM quinidine was greater in P388/ADM K562/VCR cells, and almost all the intracellular DAU was lost cells than in other resistant tumor cells. from P388/ADM cells. Addition of 3 or 10 MM quinidine to the Quinidine slightly enhanced cellular uptake of VCR and DAU culture effectively decreased this efflux of VCR or DAU from the in sensitive P388 and K562 cells. One million P388 cells accu cells. After incubation with quinidine for 1 hr, approximately 50 mulated 0.38 and 10.0 pmol of [3H]VCR and [3H]DAU, respec to 70% of the initial amount of VCR still remained in the cells. tively, and one million K562 cells accumulated 0.65 pmol of [3H]- Moreover, 50 to 65% of the initial intracellular DAU still remained in P388/ADM cells after reincubation with quinidine for 10 min, â€”100 while in the absence of quinidine, more than 90% of the drug was transported from the cells during this incubation period. Effects of Quinidine-related Compounds on VCR Cytotox- icity. In studies on the mechanism of the effect of quinidine, we examined the effects of other drugs with an antiarrhythmic action. At noncytotoxic and maximum effective concentrations, antiarrhythmic drugs in general enhanced the cytotoxicities of VCR and ADM, especially in VCR- and ADM-resistant tumor cells, but their effects were significantly lower than those of quinidine (Table 4). Of the compounds tested, lidocaine, propran- i O 1 20 .- Time ( hr ) olol, and ajimalin possess a rather strong effect, especially in Charts. Effects of quinidine on the extracellular transports of [3H]VCR and P388/VCR cells, causing a 4- to 17-fold enhancement. Similar [3H]DAU. P388/VCR (/4), K562/VCR (B), or P388/ADM (C) cells (each 2 x HP/ml medium) were incubated in glucose-free growth medium supplemented with 10% enhancement was also observed in K562/VCR cells (data not dialyzed fetal bovine serum with 10 mw sodium azide for 10 min. Then, 33 nw shown). In P388/ADM cells, propranolol and ajimalin enhanced [3H]VCR (specific activity, 5.8 Ci/mmol) or [3H]DAU (specific activity, 4.34 Ci/mmol) the cytotoxicity of ADM severalfold. was added, and incubation was continued for 30 min. Then, each cell suspension was centrifuged, and precipitated cells were resuspended and incubated at 37Â°in growth medium in the absence or presence of quinidine. At the indicated times, the intracellular [3H]VCR and [3H]DAU levels were determined as described previ DISCUSSION ously (28, 29). Release of [3H]VCR (A and B) or [3H]DAU (C) in the absence (â€¢)or presence of quinidine at 3 JIM(O) or 10 UM(A) was measured. The initial intracellular We have reported that calcium channel blockers and calmod- levels of drug in A to C were 0.587 Â±0.020, 0.717 Â±0.071, and 91.79 Â±7.92 ulin inhibitors efficiently inhibited drug efflux from resistant tumor pmol/106 cells, respectively. Points, mean for triplicate experiments. The signifi cance of differences of values with quinidine from the corresponding values without cells (24, 25, 29, 30), thereby endowing these cells with the quinidine (*, p < 0.05) was analyzed by the i test. same sensitivity to antitumor agents as sensitive cells (24-27,

Table 4 Enhancement of cytotoxicities of VCR and ADM by antiarrhythmic agents Tumor cells were exposed to graded concentrations of VCR (or ADM) with or without antiarrhythmic agents 5 hr after seeding the cells at 2 x 10* (P388 and P388/ADM) or 3 x 104 (P388/VCR) per 2 ml of medium. Cell numbers were counted 72 hr after continuous exposure to drugs, and the ICW,was determined as shown in Chart 1 (26, 29). In the absence of drugs, cells grew exponentially, and the final cell numbers of P388, P388/ VCR, P388, and P388/ADM were (2.45 Â±0.10) x 105, (2.59 Â±0.05) x 105, (2.73 Â±0.04) x 105, and (2.75 Â± 0.04) x 105 per 2 ml of medium, respectively. Each antiarrhythmic agent alone had no cytotoxicity at the concentration used. againstAntiarrhythmic 1CÂ»(riM)of VCR againstP38817.3(nw) of ADM

agentNone 78 Â±0.09a Â±1.47 Â±1.15 Â±80 Procainamide(100)l> 22.2 Â±0.16Â° 1.62 Â±0.07 17.3 Â±0.43 445 Â±30.9 0.71 Â±0.02C 7.21 Â±0.67C Lidocaine (100) 21 .6 Â±1.25 713 Â±16.3 0.94 Â±0.03Â° 11.1 Â±1.13" Diphenylhydantoin (100) 13.3 Â±1.17 548 Â±83.8 0.57 Â±0.01 c 6.27 Â±0.09C 274 Â± 6.18C Propranolol (10) 17.3Â± 1.67 0.49 Â±0.08CP388/VCR28.81.60 Â±0.42Â°ICÂ» 10.2Â±1.02CP388/ADM713193 Â±11.0Â° Ajimalin (100)P3881. * Mean Â±S.D. for 3 determinations. " Numbers in parentheses, concentration of antiantiythmic agent (/Â¿M). c Significant difference (p < 0.05) by Student's t test from the value without antiarrhythmic agent in the same cell line.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. Overcoming Drug Resistance with Quinidine 29-32). These results suggest that cellular calcium and calmod- studies. Cancer Res., 30: 1174-1184,1970. 6. DaÃ±o, K. Cross-resistance between Vinca alkaloids and anthracyclines in ulin may be related to the drug efflux mechanism in the plasma Ehrlich asertes tumor in vivo. Cancer Chemother. Rep. Part I, 56: 701-708 membrane of the resistant tumor cells. In this paper, we found 1972. that quinidine, an antiarrhythmic drug, possesses the ability to 7. Dane, K. Active outward transport of daunomycin in resistant Ehrlich asertes tumor cells. Biochim. Biophys. Acta, 323: 466-483,1973. enhance the cytotoxicity of VCR and ADM in resistant tumor 8. Fishman, M. C., and Specter, J. Potassium current suppression by quinidine cells, although its effects in vitro are considerably lower than reveals additional calcium currents in neuroblastoma cells. Proc. Nati. Acad. those of calcium channel blockers and calmodulin inhibitors (26, Sci. USA, 78: 5245-5249,1981. 9. Fuchs, F., Gertz, E. W., and Briggs, F. N. The effect of quinidine on calcium 29, 31). Potentiation of VCR and ADM effects by quinidine in accumulation by isolated sarcoplasmic reticulum of skeletal and cardiac mus vivo was also lower than that observed by calcium channel cle. J. Gen. Physiol., 52: 955-968,1968. blockers (26, 29); however, the effect of quinidine in vivo was 10. Harrow, J. A. C., and Dhalla, N. S. Effects of quinidine on calcium transport activities of the rabbit heart mitochondria and sarcotubular vesicles. Biochem. rather stronger than that observed for calmodulin inhibitors (32), Pharmacol., 25: 897-902,1976. because calmodulin inhibitors usually were toxic in animal ex 11. Hondeghem, L. M., and Mason, J. W. Agents used in cardiac arrhythmias. In: B. M. Katzung (ed.), Basic and Clinical Pharmacology, pp. 138-154. Los Altos, periments. CA: Lange Medical Publications, 1982. Calcium channel blockers inhibit the slow calcium channel in 12. Inaba, M., Kobayashi, H., Sakurai, Y., and Johnson, R. K. Active efflux of the plasma membrane of smooth muscle. Quinidine has no daunomycin and Adriamycin in sensitive and resistant sublines of P388 leu kemia. Cancer Res., 39: 2200-2203,1979. calcium channel blocking action. Quinidine and other antiarrhyth 13. Johnson, R. K., Chitnis, M. P., Embrey, W. M., and Gregory, E. B. Character mic drugs perturb the organization of membrane lipids and affect istics in vivo of resistance and cross-resistance of an Adriamycin-resistant nonspecifically various membrane-bound enzymes which are subline of P388 leukemia. Cancer Treat. Rep., 62:1535-1547,1978. 14. Juliano, R. L., and Ling, V. A surface glycoprotein modulating drug permeability believed to be influenced by the physical state of surrounding in Chinese hamster ovary cell mutant. Biochim. Biophys. Acta, 455:152-162, lipids (22). Quinidine depresses the lipid-phase transition tem 1976. 15. Lew, V. L., and Ferreira, H. G. Variable Ca sensitivity of a K-setective channel perature and broadens the transition (22). This interaction of in intact red-cell membranes. Nature (Lond.), 263: 336-338,1976. quinidine with phospholipid is suggested to contribute the mech 16. Ling, V., and Thompson, L. H. Reduced permeability in CHO cells as a anisms by which quinidine exerts its physiological and pharma mechanism of resistance to colchidne. J. Cell. Physiol., 83:103-116,1974. 17. Lozzio, C. B., and Lozzio. B. B. Human chronic myelogenous leukemia cell cological effects reported previously (1, 8, 10, 11, 15, 22). line with positive Philadelphia chromosome. Blood, 45: 321-334,1975. Calcium channel blockers are also known to interact with the 18. Nishimura, T., Suzuki, H., Mute, K., and Tanaka, N. Mechanism of Adriamycin resistance in a subline of mouse lymphoblastoma L5178Y cells. J. Antibiot. plasma membrane (2, 23, 34). As a mechanism of overcoming (Tokyo), 32: 518-522,1979. drug resistance by these agents, we must consider the possibility 19. Riordan, J. R., and Ling, V. Purification of P-glycoprotein from plasma mem that the interaction of calcium channel blockers and quinidine brane vesicles of Chinese hamster ovary cell mutants with reduced colchicine permeability. J. Biol. Chem., 254:12701-12705,1979. with the membrane of tumor cells might lead to an inhibition of 20. Skovsgaard, T. Mechanisms of resistance to daunombran in Ehrlich asertes drug efflux from resistant tumor cells. tumor cells. Cancer Res., 38: 1785-1791,1978. Another feature of quinidine is that the drug depresses my- 21. Skovsgaard, T. Mechanisms of cross-resistance between vincristine and dau- norubicin in Ehrlich asertes tumor cells. Cancer Res., 38: 4722-4727,1978. ocardiac contractility (9, 10). The real mechanism of this action 22. Surewicz, W. K., and Jozwiak, Z. Effect of quinidine on membrane properties. is not known; however, the drug has been reported to decrease Depression of the lipid phase transition temperature and changes in the permeability of the lipid bilayer. Biochem. Pharmacol., 32:1467-1471,1983. microsomal and mitochondrial calcium uptake in cardiac muscle 23. Triggle, D. J. Calcium antagonists: basic chemical and pharmacological as (10). A decrease in calcium uptake in heart subcellular particles pects. In: G. B. Weiss (ed.), New Perspectives on Calcium Antagonists, pp. can be conceived to reduce the intracellular calcium stores. Thus, 1-18. Bethesda: American Physiological Society, 1981. 24. Tsuruo. T. Reversal of acquired resistance to Vinca alkaloids and anthracycline both calcium channel blocker and quinidine can induce similar antibiotics. Cancer Treat. Rep., 67: 889-894, 1983. results, i.e., a decrease in the intracellular level of free calcium, 25. Tsuruo, T., lida, H., Naganuma, K., Tsukagoshi, S., and Sakurai, Y. Promotion although the processes are different from each other (10). by verapamil of vicristine responsiveness in tumor cell lines inherently resistant to the drugs. Cancer Res., 43: 808-813,1983. The present results are interesting in cancer chemotherapy, 26. Tsuruo, T., lida, H., Nojiri, M., Tsukagoshi, S., and Sakurai, Y. Circumvention and the mechanisms that underlie these phenomena are worth of vincristine and Adriamycin resistance in vitro and in vivo by calcium influx btockers. Cancer Res., 43: 2905-2910,1983. being elucidated. Interaction of calcium channel blockers and 27. Tsuruo, T., lida, H., Nojiri, M., Tsukagoshi, S.,and Sakurai, Y. Potentiation of quinidine with membrane and the modulation of the calcium chemotherapeutic effect of vincristine in vincristine resistant tumor bearing environment which was induced in the membrane might be mice by calmodulin inhibitor clomipramine. J. Pharmacobio-Dyn., 6:145-147, 1983. important in the inhibition of drug efflux of resistant tumor cells. 28. Tsuruo, T., lida, H., Ohkochi, E., Tsukagoshi, S., and Sakurai, Y. Establishment and properties of vincristine-resistant human myelogenous leukemia K562. Gann, 74:751-758,1983. ACKNOWLEDGMENTS 29. Tsuruo, T., lida, H., Tsukagoshi, S., and Sakurai, Y. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity We thank Shionogi and Co. for a gift of VCR. We are indebted to Elizabeth of vincristine and vinblastine by verapamil. Cancer Res., 41:1967-1972,1981. Ichihara and Mie Shimizu for editing and typing the manuscript. 30. Tsuruo, T., lida, H., Tsukagoshi, S., and Sakurai, Y. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. Effects of Quinidine and Related Compounds on Cytotoxicity and Cellular Accumulation of Vincristine and Adriamycin in Drugresistant Tumor Cells

Takashi Tsuruo, Harumi Iida, Yoshiko Kitatani, et al.

Cancer Res 1984;44:4303-4307.

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