Cancer Letters, 50 (1990:145-51
Ehvier
45
Scientific Publishers Ireland Ltd.
Novel screening method for agents that overcome classical multidrug resistance in a human cell line A. Yoshimura,
N. Shudo,
S.-I. Ikeda, M. Ichikawa, T. Sumizawa
Cancer Research Institute, Faculty of Medicine,
Kagoshimo
University,
Usuki-cho,
and S.-I. Akiyama
Kagoshima,
890 (Japan)
(Received 30 September 1989) (Accepted 27 October 1989)
Summary P-glycoprotein
(P-gp)
is inoolued in the of organic compounds including a fluorescent dye, rhodamine 6G (RG), as well as anti-cancer drugs. Agents that overcome classical multidrugresistance (MDR) increased the accumulation of RG in an MDR cell line, KB-Cl. The effect of agents on RG-accumulation in KB-Cl cells was highly correlated with their effect on the reversion of oincristine-resistance in KB-Cl cells. RG was detected on a fluorescence
transport of a wide variety
microplate reader with a rhodamine channel. This assay may become a useful method for the screening of agents that ouercome classical MBR, since it is quic.k and simple.
Keywords:
multidrug-resistance; fluorescent dye; screening method; resistance modifiers; KB cell line. Introduction
Drug resistance is a major obstacle to the chemotherapy of human cancer. One possible cause of MDR in some human tumor cell lines is the decreased accumulation of a variety of Correspondence Abbreviations;
to: S-I.
Akiyama.
MTT,3-(4,5~~dimethylthtazol-2-yl)-2,5-diphenyl-
tetrazoliumbromide;
03O4-3835/90/$03.50
AZP, azidopine.
chemically and functionally unrelated drugs. Over-expression of a membrane glycoprotein P-gp has been widely observed in various multidrug resistant cell lines. P-gp has been thought to be a pump molecule that transports anti-cancer agents outside the cells [5,12,26]. The gene encoding P-gp is called mdr-I, and MDR in which P-gp and mdr-I are involved is called classical MDR. Since recent studies have shown that P-gpassociated MDR occurs clinically [811,13,15,25], strategies designed either to block expression or to circumvent this form of drug resistance are needed. Agents that inhibit P-gp activity at concentrations with little or no cytotoxic effect may be useful in overcoming MDR in clinical chemotherapy when they are administered in combination with anti-cancer drugs. Numerous compounds have been reported to overcome MDR. They can be divided into the three groups [5]; (1) calcium channel blockers such as verapamil [22] and calmodulin inhibitors, (2) non-cytotoxic analogues of anthracyclines and vinca alkaloids and (3) other compounds that do not clearly belong to the above groups [26,28]. Recently, several dihydropyridine analogs have been shown to have a modulating effect on MDR [16,20,23]. However, most of such agents overcome MDR at the 10-5-106 M level. What is needed is to find or synthesize new agents that overcome MDR at much lower concentrations.
G 1990 Elsevier Scientific Publishers Ireland Ltd
Published and Printed in Ireland
46
The following parameters have been used to screen agents that overcome MDR: (1) enhanced cytotoxicity of anti-cancer drugs to MDR cells, (2) enhanced accumulation of anticancer drugs in MDR cells, (3) inhibition of photoaffinity labeling of P-gp with photoanalogs of anti-cancer drugs [2,3] and resistance modifiers [16,19] and (4) inhibition of binding of vinblastine or vincristine to membrane vesicles from MDR cells [7,17]. However, these methods either take a long time or need radiolabeled compounds. Willingham et al. [27] reported that the fluorescence of daunomycin can be used to measure its nuclear accumulation. They found that agents that overcome MDR such as verapamil increased the nuclear accumulation of daunomycin. Neyfakh [ 18) reported that several fluorescent dyes accumulated less in MDR cells than in drug-sensitive cells. They used fluorescence microscopy to detect accumulated dyes or drugs in the cells. However, their methods are not suitable for quantitative analysis or for the rapid screening of hundreds of compounds. Among several fluorescent basic dyes, we found that the accumulation of rhodamine-6G (RG) in KB-Cl cells was less than 10% of that in KB cells and that RG was most easily quantified on the rhodamine channel of a fluorescence microplate reader. We applied this phenomenon to the screening of the agents that overcome MDR. It seems that agents that inhibit the transport of RG by P-gp also inhibit the transport of anti-cancer drugs by P-gp and that they overcome MDR. Materials and methods Cells
A human KB epidermal carcinoma cell line was cultured in a minimal essential medium (MEM) containing 10% newborn calf serum (NCS). The multidrug resistant clone, KB-Cl was isolated from KB-3- 1 [ 11 with increasing concentrations of colchicine in the selection medium and maintained in an MEM containing 1 pg/ml colchicine. The cells were used for experiments after culturing in drug free
medium for 2 days. Relative resistance of KBCl cells to various anti-cancer drugs was described previously [ 161. Assay for celf-accumuiation of RG Confluent monolayers of KB and KB-Cl cells in 24-well or 96-well microplate dishes were incubated with 5 PM RG for indicated periods at 37OC. After washing with ice-cold phosphate buffered saline (PBS) three times, the cells were solubilized in 0.1% Triton X-100 in 10 mM phosphate buffer (pH 7.4). The fluorescence of RG (emission at 560 nm and excitation at 530 nm) was measured on a Hitachi 204 fluorescence-spectrophotometer (for 24well dishes) and a Flow Titertek* Fluorskan II with rhodamine channel (for 96-well microplates). Photoaffinity labeling of P-gp with FHlazidopine (AZP) Preparation of membrane vesicles from KB and KB-Cl cells, photoaffinity labeling with [3H]AZP in the presence of agents, and electrophoresis were carried out as described previously [ 161. for drug-sensitivity of KB and KIWI cells The effect of agents on the vincristine sensitivity of the cells was studied with an MTTassay [6]. The cells were plated at 3000 per well in 96-well microplate dishes in the presence of various concentrations of vincristine and 10 PM of several agents, and cultured for 5 days. Then the cells were incubated with 0.2 mg/ml MTT in MEM for 4 h, and the formazan was dissolved in dimethyl sulfoxide and measured as its absorbance at 570 nm. The relative resistance of KB and KB-Cl cells as compared with KB cells without agents was calculated from their IC, (the concentration that inhibits cell growth by 50%). Assay
Results Transport of RG in KB-Cl cells by P-gp We found that RG accumulated less in KBCl cells than in KB cells and that agents that
47
overcome MDR enhanced the RG accumulation. Figure 1 shows the time course of RG accumulation in KB and KB-Cl cells in the presence and absence of 20 FM cepharanthine (Cep) and 20 FM reserpine (Res). The accumulation of RG in KB cells reached a plateau within 30 min and was rarely affected by cepharanthine and reserpine. The accumulation of RG in KB-Cl was about 10% of that in KB cells in the absence of agent3 and 5075% in their presence. After 90 min of incubation, the medium was removed and then fresh MEM (without RG and agents) was added. Cell-associated RG in KB cells decreased to 75% of the initial level after 50 min. RG in KBCl decreased more rapidly than in KB cells and was 5% of initial level after 50 min. These data suggest rapid :RG efflux from KB-C 1 cells. This efflux was completely inhibited by reserpine and cepharamhine (data not shown).
I ,.q
I
1wash j-+-y
1. 0
0.6 0.6
0.2
0
30
HI
90 lime
Fig. 1.
To examine whether RG interacts with Pgp, membrane vesicles from KB-Cl cells were photolabeled with [3H]AZP in the presence of RG and other agents that overcome MDR. As shown in Fig. 2, a 170-kDa protein (P-gp) in KB-Cl vesicles (lane 2) but not in KB vesicles (lane 1) was photolabeled with [3H]AZP. Cepharanthine and reserpine effectively inhibited the photolabeling of P-gp (lanes 6,7). RG also inhibited photolabeling partially at 10 FM and completely at 50-100 FM (lanes 3-5). These data suggest that RG is transported by P-gp in KB-Cl cells and that the transport was inhibited by the agents that overcome MDR.
120
150
( min)
The time course of RG accumulation and efflux in KB (closed symbol) and KB-Cl cells (open symbol), and the effect of quinidine and reserpine. Confluent monolayers of KB cells and KB-Cl cells grown in 24-well dishes were incubated with 5 PM RG in the absence (O,*) and presence of 20 PM cepharanthine (Cep; q,W) and reserpine (Res; A,A) for indicated periods. After incubation for 90 min, the medium was changed to a fresh one without any agents, and the cells were further incubated for the indicated periods. Cell-associated RG was measured with a fluorescence spectrophotometer.
RG-accumulation assay for screening of agents that overcome MDR Figure 3 shows the effect of agents that overcome MDR on RG accumulation in KB and KB-Cl cells grown in 96-well microplates. Cell-associated RG was measured with a fluorescence microplate reader. Cepharanthine, reserpine, quinidine and verapamil, which overcome MDR efficiently [14,21,22,24], increased RG accumulation in KB-Cl cells. They rarely affected RG accumulation in KB cells. We tested the effect of previously described dihydropyridine analogs [lb] on RG accumulation in KB-Cl cells. The three compounds in Group I (PAK-2,3,4) almost completely overcame the resistance to vincristine in KB-Cl cells. Those in Group II (PAK-5,6,7) moderately effected or overcame MDR, and those in Group III (PAK-8,9,10) had little effect [16]. As is shown in Fig. 4, all three compounds in Group I effectively increased the RG-accumulation in KB-Cl cells; the concentration of compounds that increased RG-accumulation in KB-Cl cells to 50% of that in KB cells was 5 -20 FM, and that in Group II was more than 50 FM. Compounds in Group III had little effect on RG accumulation. These data give further support to the hypothesis that the ability of dihydropyridine analogs to overcome MDR is highly correlated to their ability to increase RG accumulation in KB-Cl cells. To demonstrate the correlation between the modulating effect on MDR and RG accumula-
MP
-
Agents Cont. (pbl)
-
RG
RG
RG
Cep
Res
10
50
100
10
10
Cell
KB-Cl
Fig. 2. Effect of RG, cepharanthine (Cep) and reserpine (Res) on photoaffinity labeling of P-gp with [3H]AZP. Membrane vesicles from KB (lane 1) and KB-Cl (lanes 2-7) were photolabeled with 0.5 FM [3H]AZP in the presence of indicated concentrations of agents. After photolabeling, samples were subjected to electrophoresis and fluorography as described previously 1161.
tion in more detail, we tested 40 dihydropyridine analogs which were synthesized by Nissan Chemicals Ind., Chiba, Japan. Ten of them had no effect on both RG accu-
I
0
.
J
100
IO
1
Reversing
Agents
(+I)
mulation and on the vincristine resistance of KB-Cl cells. The correlations in the other 30 compounds between RG accumulation and the modulating effect on vincristine resistance are shown in Fig. 5. The coefficient of correlation value (r) was 0.80 (P < 0.01). Six compounds that increased RG-uptake in KB-Cl cells to 50% of that in KB cells at less than 5 FM showed especially strong overcoming
Fig. 3. Effect of agents that overcome MDR on RG accumulation. Accumulation of RG in KB cells (closed symbol) and KB-Cl cells (open symbol) was measured in the presence of indicated concentrations of quinidine reserpine (Res; V,V), cepharanthine (Qun; qW, (Cep; 0.0) and verapamil (Ver; A, A). Cells were incubated with 5 PM RG and indicated concentrations of chemicals for 1 h at 37T. After cells were washed with cold PBS, cell-associated RG levels were measured with a fluorescence microplate reader. A.U.: arbitrary unit.
49
0
WK-4 F#K-3
l
. . /
.
PAK-2
PAK-5
6
PAIL6 PAK-‘I
4
2
i- = 0.60 0
2
100
IO Dihydropyridine
Analogs
OJM)
Fig. 4.
Effect of dihydropridine analogs on the accumulation of RG in KB-Cl cells. Cells were incubated with 5 PM RG and indicated concentrations of compounds for 1 h at 37OC. After washing with cold PBS, cell-associated RG levels were measured as described in Fig. 3.
effects on vincristine resistance. These compounds completely overcame not only vincristine resistance but also Adriamycin resistance in KB-Cl cells at 5 ktM (data not shown). In conclusion, w’e found a high correlation between the ability of compounds to enhance RG accumulation and to overcome MDR. We expect our microplate assay to be of use in the screening of agents that overcome MDR. Discussion In this study, we established a new method for the screening Iof agents that overcome classical MDR. This method is carried out in 96-well microplates, takes only 2 h, and needs no radioactive compounds. Here we have only presented quantitative data from a fluorescence microplate reader, but RG accumulation can be directly visualized with UV illumination. This method is suitable for the first screening to find inhibitors of P-gp from hundreds of compounds.
2
K
IO
RG-ECsa
(+I)
Fig. 5. Correlation between the ability of dihydropyridine analogs to increase RG accumulation and to overcome vincistine resistance in KB-Cl cells. The effect of 40 newly synthesized dihydropyridine analogs on RG accumulation was measured as described in Fig. 3. RG accumulation in KB cells was 10.0 A.U., and EC, (the effective concentration that enhances RG accumulation to 50% of that in KB cells) of compounds in KB-Cl cells is represented by the abscissa. IC, of vincristine in KBCl cells in the presence of compounds is represented by the ordinate. Data are given for 30 effective compounds.
After further screening in vitro and in vivo, the compounds that overcome MDR may be used in clinical application together with anticancer drugs. P-gp has been shown to be responsible for intrinsic drug resistance in some types of human cancer such as renal cell carcinoma, colon carcinoma and hepatoma [ 11,151. The agents that overcome MDR might be useful for the chemotherapy of such naturally drug-resistant tumors. Goldstein et al. [ 111 and Holmes et al. [ 131 suggest that P-gp is frequently detected in tumor at the clinically refractory state. Their data suggest that P-gp is also involved in acquired drug-resistance. Dalton et al. [8] used verapamil in conjugation with anti-cancer drugs in myeloma and lymphoma patients who were P-gp positive, and
50
they reported that verapamil partially circumvents drug resistance in these patients. These studies show the clinical importance of the agents that overcome MDR. Our new method may be valid to find MDR-overcoming agents with less side effects among a large number of candidates.
11
Acknowledgments
12
We thank Dr. Sakoda and Dr. Seto (Nissan Chemicals Ind.) for their generous gift of a series of dihydropyridine analogs.
13
References
14
1
2
3
4
5
6
7
8
9
10
Akiyama, S., Fojo, A., Hanover, J.A., Pastan, I. and Gottesman, M.M. (1985) Isolation and genetic characterization of human KE cells resistant to multiple drugs. Somat. Cell. Mol. Genet., 11, 117-126. Akiyama, S., Cornwell, M.M., Kuwano, M., Pastan, I. and Gottesman, M.M. (1988) Most drugs that reverse multidrug resistance also inhibit photoaffinity labeling of P-glycoprotein by a vinblastine analog. Mol. Pharmacol., 33, 144-147. Beck, W.T.. Cirtain, M.C., Glover, C.J., Felsted, R.L. and Safa, A.R. (1988) Effect of indole alkaloids on multidrug resistance and labeling of P-glycoprotein by a photoaffinity analog of vinblastine. Biochem. Biophys. Res. Comm., 153,959-966. Bell, D.R., Gerlach, J.H., Kartner, N., Buick, R.N. and Ling, V. (1985) Detection of P-glycoprotein in ovarian cancer: a molecular marker associated with multidrug resistance. J. Clin. Oncol., 3, 311-315. Bradley, G., Juranka, P.F. and Ling, V. (1988) Mechanism of multidrug resistance. Biochim. Biophys. Acta, 948,87-128. Carmichael, J. DeGraff, W.G., Gazdar, A.F., Minna, J.D. and Mitchell, J.B. (1987) Evaluation of a tetrazoliumbased semiautomated calorimetric assay: assessment of chemosensitivity testing. Cancer Res., 47,936-942. Cornwell, M.M., Gottesman, M.M. and Pastan, I (1986) Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells. J. Biol. Chem., 261, 79217928. Dalton, W.S., Grogan, T.M., Meltzer, P.S., Scheper, R.J., Durie, B.G.M., Taylor, C.W., Miller, T.P. and Salmon, S.E. (1989) Drug-resistance in multiple myeloma and non-Hodgkin’s lymphoma: detection of P-glycoprotein and potential circumvension by addition of verapamil to chemotherapy. J. Clin. Oncol., 7.415-424. Fojo, A.T., Ueda, K., Slamon, D.J., Poplack, D.G., Gottesman, M.M. and Pastan, I. (1989) Expression of a multidrug resistance gene in human tumors and tissues. Proc. Natl. Acad. Sci. USA, 84, 265-269. Gerlach. J.H., Bell, D.R., Karakousis, C., Slocum, H.K.,
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Kartner, N., Rustum, Y.M., Ling, V. and Baker, R.M. (1987) P-glycoprotein in human sarcoma: evidence for multidrug resistance. J. Clin. Oncol., 5, 1452- 1460. Goldstein, L.J., Galski, H., Fojo, A., Willingham, M., Lai, S.-L., Gazdar, A., Pirker, R., Green, A., Crist, W., Brodeur, G.M., Lieber, M., Cossman, J., Gottesman, M.M. and Pastan, I. (1989) Expression of a multidrug resistance gene in human cancers. J. Natl. Cancer. Inst., 81, 116124. Gottesman, M.M. and Pastan, I. (1988) The multidrug transporter, a double-edged sword. J. Biol. Chem., 263, 12163-12166. Holmes, J.H., Jacobs, A., Carter, G., Wieczorek, A.J. and Padua, R.A. (1989) Multidrug resistance in haemopoietic cell lines, myelodysplastic syndromes and acute myeoblastic leukemia. Br. J. Haematol., 72,40-44. Inaba, M., Fujikura, R., Tsukagoshi, S. and Sakurai, Y. (1981) Restored in vitro sensitivity of Adriamycin”and vincristine-resistant P388 leukemia with reserpine. Biochem. Pharmacol., 30, 2191-2194. Kakehi, Y., Kanamaru, H., Yoshida, O., Ohkubo, H., Nakanishi, S., Gottesman, M.M., and Pastan, I. (1988) Measurement of mutlidrug-resistance messenger RNA in urogenital cancers; elevated expression in renal cell carcinoma is associated with intrinsic drug resistance. J. Ural., 139,862-865. Kamiwatari, K., Nagata, Y ., Kikuchi, H., Yoshimura, A., Sumizawa, T., Shudo, N., Sakoda, R., Seto, K. and Akiyama, S. (1989) Correlation between reversing of multidrug resistance inhibiting of and PHjazidopine photolabeling of P-glycoprotein of newly synthesized dihydropyrine analogs. Cancer Res., 49.3190-3195. Naito, M., Hamada. H. and Tsuruo, T. (1988) ATP/Mg*+ dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells. J. Blol. Chem., 263, 11887-11891. Neyfakh, A.A. (1988) Use of fluorescent dyes as molecular probes for the study of multidrug resistance. Exp. Cell Res., 174,168-176. Safa, A.R., Glover, C.J., Sewell, J.L., Meyers, M.B., Biedler, J.L. and Felsted, R.L. (1987) Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for calcium channel blockers. J. Biol. Chem., 262,7884-7888. Shinoda, H., Inaba, M. and Tsuruo, T. (1989) In vitro circumvention of vincristine resistance in mice with P388 leukemia using a novel compound, AHC-52. Cancer Res., 49,1722-1726. Shiraishi, N., Akiyama, S., Nakagawa, M., Kobayashi. M. and Kuwano, M. (1987) Effect of bisbenzylisoquinoline (Biscoclaurine) alkaloids on multidrug resistance in KB human cancer cells. Cancer Res., 47,2413-2416. Tsuruo, T., Iida, H., Tsukagoshi, S. and Sakurai, Y. (1981) Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res., 41 1967- 1972. Tsuruo,
T., lida, H., Nojiri, M., Tsukagoshi,
S. and Saku-
51
24
25
rai, Y. (1983) Circumvention of vincristine and Adriamycin resistance in vitro and in vivo by calcium influx blockers, Cancer Res., 43,2905-2910.
26
Tsuruo, T., lida, H., Kitatani, Y ., Yokota, K., Tsukagoshi, S. and Sakurai, Y. 11984) Effect of quinidine and related compounds on cytctoxicity and cellular accumulation of vincristine and Adriamycin in drug-resistant tumor cells.
27
Cancer Res., 44, 14l- 148. Tsuruo, T., Sugimoto, Y., Hamada, H., Roninson, I., Okumura, M., Adachi, K., Morishima, Y. and Ohno, R. (1987) Detection of multidrug resistance markers, P-glycoprotein and mdrl mRNA, in human leukemia cells. Jpn. J. Cancer. Res. (Gann), 78, 1415-1419.
28
Tsuruo, T. (1988) Mechanisms of multidrug resistance and implications for therapy. Jpn. J. Cancer Res. (Gann), 79, 285-296. Willingham, M.C., Cornwell, M.M., Cardarelli, C.O., Gottesman, M.M. and Pastan, 1. (1986) Single cell analysis of daunomycin uptake and efflux in multidrug-resistant and -sensitive KB cells: effects of verapamil and other drugs. Cancer Res., 46,5941-5946. Zamora, J.M., Pearce, H.L. and Beck, W.T. (1988) Physical-chemical properties shared by compounds that modulate multidrug resitance in human leukemic cells. Mol. Pharmacol., 33,454-462.
Ehvier
45
Scientific Publishers Ireland Ltd.
Novel screening method for agents that overcome classical multidrug resistance in a human cell line A. Yoshimura,
N. Shudo,
S.-I. Ikeda, M. Ichikawa, T. Sumizawa
Cancer Research Institute, Faculty of Medicine,
Kagoshimo
University,
Usuki-cho,
and S.-I. Akiyama
Kagoshima,
890 (Japan)
(Received 30 September 1989) (Accepted 27 October 1989)
Summary P-glycoprotein
(P-gp)
is inoolued in the of organic compounds including a fluorescent dye, rhodamine 6G (RG), as well as anti-cancer drugs. Agents that overcome classical multidrugresistance (MDR) increased the accumulation of RG in an MDR cell line, KB-Cl. The effect of agents on RG-accumulation in KB-Cl cells was highly correlated with their effect on the reversion of oincristine-resistance in KB-Cl cells. RG was detected on a fluorescence
transport of a wide variety
microplate reader with a rhodamine channel. This assay may become a useful method for the screening of agents that ouercome classical MBR, since it is quic.k and simple.
Keywords:
multidrug-resistance; fluorescent dye; screening method; resistance modifiers; KB cell line. Introduction
Drug resistance is a major obstacle to the chemotherapy of human cancer. One possible cause of MDR in some human tumor cell lines is the decreased accumulation of a variety of Correspondence Abbreviations;
to: S-I.
Akiyama.
MTT,3-(4,5~~dimethylthtazol-2-yl)-2,5-diphenyl-
tetrazoliumbromide;
03O4-3835/90/$03.50
AZP, azidopine.
chemically and functionally unrelated drugs. Over-expression of a membrane glycoprotein P-gp has been widely observed in various multidrug resistant cell lines. P-gp has been thought to be a pump molecule that transports anti-cancer agents outside the cells [5,12,26]. The gene encoding P-gp is called mdr-I, and MDR in which P-gp and mdr-I are involved is called classical MDR. Since recent studies have shown that P-gpassociated MDR occurs clinically [811,13,15,25], strategies designed either to block expression or to circumvent this form of drug resistance are needed. Agents that inhibit P-gp activity at concentrations with little or no cytotoxic effect may be useful in overcoming MDR in clinical chemotherapy when they are administered in combination with anti-cancer drugs. Numerous compounds have been reported to overcome MDR. They can be divided into the three groups [5]; (1) calcium channel blockers such as verapamil [22] and calmodulin inhibitors, (2) non-cytotoxic analogues of anthracyclines and vinca alkaloids and (3) other compounds that do not clearly belong to the above groups [26,28]. Recently, several dihydropyridine analogs have been shown to have a modulating effect on MDR [16,20,23]. However, most of such agents overcome MDR at the 10-5-106 M level. What is needed is to find or synthesize new agents that overcome MDR at much lower concentrations.
G 1990 Elsevier Scientific Publishers Ireland Ltd
Published and Printed in Ireland
46
The following parameters have been used to screen agents that overcome MDR: (1) enhanced cytotoxicity of anti-cancer drugs to MDR cells, (2) enhanced accumulation of anticancer drugs in MDR cells, (3) inhibition of photoaffinity labeling of P-gp with photoanalogs of anti-cancer drugs [2,3] and resistance modifiers [16,19] and (4) inhibition of binding of vinblastine or vincristine to membrane vesicles from MDR cells [7,17]. However, these methods either take a long time or need radiolabeled compounds. Willingham et al. [27] reported that the fluorescence of daunomycin can be used to measure its nuclear accumulation. They found that agents that overcome MDR such as verapamil increased the nuclear accumulation of daunomycin. Neyfakh [ 18) reported that several fluorescent dyes accumulated less in MDR cells than in drug-sensitive cells. They used fluorescence microscopy to detect accumulated dyes or drugs in the cells. However, their methods are not suitable for quantitative analysis or for the rapid screening of hundreds of compounds. Among several fluorescent basic dyes, we found that the accumulation of rhodamine-6G (RG) in KB-Cl cells was less than 10% of that in KB cells and that RG was most easily quantified on the rhodamine channel of a fluorescence microplate reader. We applied this phenomenon to the screening of the agents that overcome MDR. It seems that agents that inhibit the transport of RG by P-gp also inhibit the transport of anti-cancer drugs by P-gp and that they overcome MDR. Materials and methods Cells
A human KB epidermal carcinoma cell line was cultured in a minimal essential medium (MEM) containing 10% newborn calf serum (NCS). The multidrug resistant clone, KB-Cl was isolated from KB-3- 1 [ 11 with increasing concentrations of colchicine in the selection medium and maintained in an MEM containing 1 pg/ml colchicine. The cells were used for experiments after culturing in drug free
medium for 2 days. Relative resistance of KBCl cells to various anti-cancer drugs was described previously [ 161. Assay for celf-accumuiation of RG Confluent monolayers of KB and KB-Cl cells in 24-well or 96-well microplate dishes were incubated with 5 PM RG for indicated periods at 37OC. After washing with ice-cold phosphate buffered saline (PBS) three times, the cells were solubilized in 0.1% Triton X-100 in 10 mM phosphate buffer (pH 7.4). The fluorescence of RG (emission at 560 nm and excitation at 530 nm) was measured on a Hitachi 204 fluorescence-spectrophotometer (for 24well dishes) and a Flow Titertek* Fluorskan II with rhodamine channel (for 96-well microplates). Photoaffinity labeling of P-gp with FHlazidopine (AZP) Preparation of membrane vesicles from KB and KB-Cl cells, photoaffinity labeling with [3H]AZP in the presence of agents, and electrophoresis were carried out as described previously [ 161. for drug-sensitivity of KB and KIWI cells The effect of agents on the vincristine sensitivity of the cells was studied with an MTTassay [6]. The cells were plated at 3000 per well in 96-well microplate dishes in the presence of various concentrations of vincristine and 10 PM of several agents, and cultured for 5 days. Then the cells were incubated with 0.2 mg/ml MTT in MEM for 4 h, and the formazan was dissolved in dimethyl sulfoxide and measured as its absorbance at 570 nm. The relative resistance of KB and KB-Cl cells as compared with KB cells without agents was calculated from their IC, (the concentration that inhibits cell growth by 50%). Assay
Results Transport of RG in KB-Cl cells by P-gp We found that RG accumulated less in KBCl cells than in KB cells and that agents that
47
overcome MDR enhanced the RG accumulation. Figure 1 shows the time course of RG accumulation in KB and KB-Cl cells in the presence and absence of 20 FM cepharanthine (Cep) and 20 FM reserpine (Res). The accumulation of RG in KB cells reached a plateau within 30 min and was rarely affected by cepharanthine and reserpine. The accumulation of RG in KB-Cl was about 10% of that in KB cells in the absence of agent3 and 5075% in their presence. After 90 min of incubation, the medium was removed and then fresh MEM (without RG and agents) was added. Cell-associated RG in KB cells decreased to 75% of the initial level after 50 min. RG in KBCl decreased more rapidly than in KB cells and was 5% of initial level after 50 min. These data suggest rapid :RG efflux from KB-C 1 cells. This efflux was completely inhibited by reserpine and cepharamhine (data not shown).
I ,.q
I
1wash j-+-y
1. 0
0.6 0.6
0.2
0
30
HI
90 lime
Fig. 1.
To examine whether RG interacts with Pgp, membrane vesicles from KB-Cl cells were photolabeled with [3H]AZP in the presence of RG and other agents that overcome MDR. As shown in Fig. 2, a 170-kDa protein (P-gp) in KB-Cl vesicles (lane 2) but not in KB vesicles (lane 1) was photolabeled with [3H]AZP. Cepharanthine and reserpine effectively inhibited the photolabeling of P-gp (lanes 6,7). RG also inhibited photolabeling partially at 10 FM and completely at 50-100 FM (lanes 3-5). These data suggest that RG is transported by P-gp in KB-Cl cells and that the transport was inhibited by the agents that overcome MDR.
120
150
( min)
The time course of RG accumulation and efflux in KB (closed symbol) and KB-Cl cells (open symbol), and the effect of quinidine and reserpine. Confluent monolayers of KB cells and KB-Cl cells grown in 24-well dishes were incubated with 5 PM RG in the absence (O,*) and presence of 20 PM cepharanthine (Cep; q,W) and reserpine (Res; A,A) for indicated periods. After incubation for 90 min, the medium was changed to a fresh one without any agents, and the cells were further incubated for the indicated periods. Cell-associated RG was measured with a fluorescence spectrophotometer.
RG-accumulation assay for screening of agents that overcome MDR Figure 3 shows the effect of agents that overcome MDR on RG accumulation in KB and KB-Cl cells grown in 96-well microplates. Cell-associated RG was measured with a fluorescence microplate reader. Cepharanthine, reserpine, quinidine and verapamil, which overcome MDR efficiently [14,21,22,24], increased RG accumulation in KB-Cl cells. They rarely affected RG accumulation in KB cells. We tested the effect of previously described dihydropyridine analogs [lb] on RG accumulation in KB-Cl cells. The three compounds in Group I (PAK-2,3,4) almost completely overcame the resistance to vincristine in KB-Cl cells. Those in Group II (PAK-5,6,7) moderately effected or overcame MDR, and those in Group III (PAK-8,9,10) had little effect [16]. As is shown in Fig. 4, all three compounds in Group I effectively increased the RG-accumulation in KB-Cl cells; the concentration of compounds that increased RG-accumulation in KB-Cl cells to 50% of that in KB cells was 5 -20 FM, and that in Group II was more than 50 FM. Compounds in Group III had little effect on RG accumulation. These data give further support to the hypothesis that the ability of dihydropyridine analogs to overcome MDR is highly correlated to their ability to increase RG accumulation in KB-Cl cells. To demonstrate the correlation between the modulating effect on MDR and RG accumula-
MP
-
Agents Cont. (pbl)
-
RG
RG
RG
Cep
Res
10
50
100
10
10
Cell
KB-Cl
Fig. 2. Effect of RG, cepharanthine (Cep) and reserpine (Res) on photoaffinity labeling of P-gp with [3H]AZP. Membrane vesicles from KB (lane 1) and KB-Cl (lanes 2-7) were photolabeled with 0.5 FM [3H]AZP in the presence of indicated concentrations of agents. After photolabeling, samples were subjected to electrophoresis and fluorography as described previously 1161.
tion in more detail, we tested 40 dihydropyridine analogs which were synthesized by Nissan Chemicals Ind., Chiba, Japan. Ten of them had no effect on both RG accu-
I
0
.
J
100
IO
1
Reversing
Agents
(+I)
mulation and on the vincristine resistance of KB-Cl cells. The correlations in the other 30 compounds between RG accumulation and the modulating effect on vincristine resistance are shown in Fig. 5. The coefficient of correlation value (r) was 0.80 (P < 0.01). Six compounds that increased RG-uptake in KB-Cl cells to 50% of that in KB cells at less than 5 FM showed especially strong overcoming
Fig. 3. Effect of agents that overcome MDR on RG accumulation. Accumulation of RG in KB cells (closed symbol) and KB-Cl cells (open symbol) was measured in the presence of indicated concentrations of quinidine reserpine (Res; V,V), cepharanthine (Qun; qW, (Cep; 0.0) and verapamil (Ver; A, A). Cells were incubated with 5 PM RG and indicated concentrations of chemicals for 1 h at 37T. After cells were washed with cold PBS, cell-associated RG levels were measured with a fluorescence microplate reader. A.U.: arbitrary unit.
49
0
WK-4 F#K-3
l
. . /
.
PAK-2
PAK-5
6
PAIL6 PAK-‘I
4
2
i- = 0.60 0
2
100
IO Dihydropyridine
Analogs
OJM)
Fig. 4.
Effect of dihydropridine analogs on the accumulation of RG in KB-Cl cells. Cells were incubated with 5 PM RG and indicated concentrations of compounds for 1 h at 37OC. After washing with cold PBS, cell-associated RG levels were measured as described in Fig. 3.
effects on vincristine resistance. These compounds completely overcame not only vincristine resistance but also Adriamycin resistance in KB-Cl cells at 5 ktM (data not shown). In conclusion, w’e found a high correlation between the ability of compounds to enhance RG accumulation and to overcome MDR. We expect our microplate assay to be of use in the screening of agents that overcome MDR. Discussion In this study, we established a new method for the screening Iof agents that overcome classical MDR. This method is carried out in 96-well microplates, takes only 2 h, and needs no radioactive compounds. Here we have only presented quantitative data from a fluorescence microplate reader, but RG accumulation can be directly visualized with UV illumination. This method is suitable for the first screening to find inhibitors of P-gp from hundreds of compounds.
2
K
IO
RG-ECsa
(+I)
Fig. 5. Correlation between the ability of dihydropyridine analogs to increase RG accumulation and to overcome vincistine resistance in KB-Cl cells. The effect of 40 newly synthesized dihydropyridine analogs on RG accumulation was measured as described in Fig. 3. RG accumulation in KB cells was 10.0 A.U., and EC, (the effective concentration that enhances RG accumulation to 50% of that in KB cells) of compounds in KB-Cl cells is represented by the abscissa. IC, of vincristine in KBCl cells in the presence of compounds is represented by the ordinate. Data are given for 30 effective compounds.
After further screening in vitro and in vivo, the compounds that overcome MDR may be used in clinical application together with anticancer drugs. P-gp has been shown to be responsible for intrinsic drug resistance in some types of human cancer such as renal cell carcinoma, colon carcinoma and hepatoma [ 11,151. The agents that overcome MDR might be useful for the chemotherapy of such naturally drug-resistant tumors. Goldstein et al. [ 111 and Holmes et al. [ 131 suggest that P-gp is frequently detected in tumor at the clinically refractory state. Their data suggest that P-gp is also involved in acquired drug-resistance. Dalton et al. [8] used verapamil in conjugation with anti-cancer drugs in myeloma and lymphoma patients who were P-gp positive, and
50
they reported that verapamil partially circumvents drug resistance in these patients. These studies show the clinical importance of the agents that overcome MDR. Our new method may be valid to find MDR-overcoming agents with less side effects among a large number of candidates.
11
Acknowledgments
12
We thank Dr. Sakoda and Dr. Seto (Nissan Chemicals Ind.) for their generous gift of a series of dihydropyridine analogs.
13
References
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