ADM) cells

Cancer Letters 177 (2002) 89–93 www.elsevier.com/locate/canlet

Inhibition of P-glycoprotein by flavonoid derivatives in adriamycin-resistant human myelogenous leukemia (K562/ADM) cells Tomomi Ikegawa a, Hisakazu Ohtani a, Noriko Koyabu a, Motoharu Juichi b, Yukiko Iwase b, Chihiro Ito c, Hiroshi Furukawa c, Mikihiko Naito d, Takashi Tsuruo d, Yasufumi Sawada a,* a

Department of Medico-Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan b Faculty of Pharmaceutical Sciences, Mukogawa Women’s University, Nishinomiya, Hyogo 663-8179, Japan c Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468-8503, Japan d Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku 113-0032, Japan Received 3 September 2001; received in revised form 5 September 2001; accepted 7 September 2001

Abstract We investigated the effects of natural flavones, quercetin and morin, and their pentamethyl, pentaethyl, pentapropyl, pentabutyl and pentaallyl ethers, on the function of P-glycoprotein (P-gp) assessed by an increase in the uptake of [ 3H]vincristine by human myelogenous leukemia (K562) cells and adriamycin-resistant human myelogenous leukemia (K562/ADM) cells. Pentamethyl, pentaethyl, pentapropyl and pentaallyl ethers of morin and quercetin (20 mM) all increased the uptake of [ 3H]vincristine by K562/ADM cells, while quercetin, morin and their pentabutyl ethers had no effect. Pentamethylquercetin, pentaallylquercetin and pentaethylmorin remarkably increased the uptake of [ 3H]vincristine by K562/ADM cells by 10.6, 10.8 and 14.4-fold, respectively. These inhibitory potencies for P-gp were more potent than typical P-gp inhibitors, cyclosporine A and verapamil. Taking into consideration that these flavonoid derivatives possess antitumor promoter activity, they may become candidates of effective multidrug resistance-reversing agents in cancer chemotherapy. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Flavonoid derivatives; P-glycoprotein; Vincristine; Multidrug resistance

1. Introduction Acquisition of multidrug resistance (MDR) in tumor cells has been one of the most significant obstacles in cancer chemotherapy [1]. MDR is, at least in part, conferred by transporters such as P-glycoprotein (P* Corresponding author. Tel.: 181-92-642-6610; fax: 181-92642-6614. E-mail address: [email protected] (Y. Sawada).

gp) which are expressed on the plasma membranes of tumor cells and actively extrude a wide variety of anticancer agents from the cells [2]. Indeed, P-gp is overexpressed in most multidrug-resistant tumor cells [3]. P-gp possesses two adenosine triphosphate- (ATP)binding cassettes and extrudes anticancer agents in an energy-dependent manner [4]. Recently, it has been reported that P-gp significantly contributes not only to MDR but also to basal sensitivity of drugnaive tumor cells to anticancer agents, even if the

0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00761-3

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expression level of P-gp is quite low. Therefore, suppression of P-gp function by P-gp inhibitor may increase the effectiveness of cancer chemotherapy even in the treatment of drug-naive tumors as well as multidrug-resistant tumors. Until now, we have searched for novel P-gp inhibitors from daily foods and beverages, and found furanocoumarin derivatives and methoxyflavones from grapefruit juice and orange juice, respectively [6,7]. Especially, methoxyflavones potently inhibit P-gpmediated transport without affecting the enzymatic activity of cytochrome P450 (CYP) 3A4 [7], which is inhibited by most of the P-gp inhibitors. We have also reported that methoxyflavones such as nobiletin, tangeretin and 3,3 0 ,4 0 ,5,6,7,8-heptamethoxyflavone increased more than ten-fold the intracellular accumulation of vincristine in adriamycin-resistant human myelogenous leukemia (K562/ADM) cells [8]. The effects of these methoxyflavones were comparable to those of well-known P-gp inhibitors, verapamil (VER) and cyclosporine A (CyA) [8]. We have also reported that polymethoxyflavones including pentamethoxymorin and pentamethoxyquercetin, and other eight flavonoid derivatives including pentaethyl ethers, pentapropyl ethers, pentabutyl ethers and pentaallyl ethers of morin and quercetin (Fig. 1) showed significant inhibitory effect on Epstein–Barr virus early antigen (EBV-EA) activation

induced by a tumor promoter, 12-O-tetradecanoylphobol 13-acetate (TPA) [9]. Among the above ten flavonoid derivatives, quercetin pentaallyl ether exhibited potent antitumor promoting effect [9]. However, it is yet unclear whether these flavonoid derivatives inhibit P-gp function. If so, the dual effects (i.e. P-gp inhibition and antitumor promoter effect) of these flavonoid derivatives may synergically act in cancer chemotherapy. The aim of this study is to investigate the effects of flavonoid derivatives on Pgp function by using human myelogenous leukemia (K562) cells and K562/ADM cells. 2. Materials and methods 2.1. Materials [ 3H]Vincristine sulfate (6.6 Ci/mmol) was purchased from Amersham International (Buckinghamshire, UK). Ten flavone derivatives were synthesized by etherification of morin or quercetin with alkylhalide/K2CO3 in refluxing dimethylformamide (DMF/acetone). VER was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). CyA was a kind gift from Novartis Pharma Inc. (Basel, Switzerland). All other reagents used were commercial products of reagent grade. 2.2. Cell culture Human myelogenous leukemia K562 cells and their adriamycin-resistant variant K562/ADM cells were grown in suspension in RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) supplemented with 10% fetal calf serum, 100 mg/ml gentamicin (Schering-Plough., Osaka, Japan) at 378C in a humidified atmosphere of 95% air and 5% CO2. K562/ ADM cells were cultured in the presence of 300 ng/ ml adriamycin (Kyowa Hakko Co., Ltd, Tokyo, Japan) once every 2 weeks and were grown in drugfree medium 1 week before the experiments. 2.3. Uptake of [ 3H]vincristine

Fig. 1. Chemical structures of morin, quercetin and their pentaetherified derivatives used in this study.

Uptake experiment was performed as described in the previous report [10]. Briefly, K562 cells and K562/ ADM cells were seeded at 1 £ 10 6 cells on four-well multidishes (Nunc., Denmark). Uptake was initiated

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by adding growth medium containing 25 nM [ 3H]vincristine in the presence or absence of one of 20 mM VER, CyA, morin, quercetin or their derivatives for 2 h at 378C. The cells were then washed twice with 1 ml of ice-cold phosphate-buffered saline (PBS) (2) to stop the uptake. After the uptake, cells were dissolved with 1 M NaOH (200 ml) and neutralized with 1 M HCl (200 ml). To assay the radiolabeled compounds, all samples were transferred into counting vials and mixed with scintillation fluid (Clearsol I; Nakalai Tesque, Kyoto, Japan), and measured with a liquid scintillation counter (LS6500; Beckman Instruments, Inc., Fullerton, CA, USA). The amount of protein in K562 and K562/ADM cells in uptake studies was measured by Lowry’s method [11]. The uptake of [ 3H]vincristine was expressed as cell/medium ratio (ml/mg protein), the ratio of uptake amount into cells (pmol/mg protein) to the concentration of [ 3H]vincristine in the medium (pmol/ml).

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K562/ADM cells were 71.5 ^ 2.38 and 5.46 ^ 0.19 ml/mg protein (mean ^ SEM, n ¼ 8), respectively. K562/ADM cells exhibited remarkable resistance. 3.2. Effects of quercetin derivatives on the uptake of [ 3H]vincristine

3. Results

Fig. 2 represents the effects of 20 mM quercetin, pentamethoxyquercetin (Q-M), quercetin pentaethyl ether (Q-E), quercetin pentapropyl ether (Q-P), quercetin pentabutyl ether (Q-B) and quercetin pentaallyl ether (Q-A) on the uptake of [ 3H]vincristine by K562 cells and K562/ADM cells, along with that of 20 mM VER and CyA. Q-M, Q-E, Q-P and Q-A significantly increased the uptake of [ 3H]vincristine by K562/ADM cells. Among the above derivatives, the effects of Q-E and Q-A were more potent than typical P-gp inhibitors, VER and CyA (Fig. 2(B)). Although the above four derivatives, VER and CyA also significantly increased the uptake of [ 3H]vincristine in K562 cells, the rates of increases were far smaller than that observed in K562/ADM cells (Fig. 2(A)).

3.1. The uptake of [ 3H]vincristine by K562 cells and K562/ADM cells

3.3. Effects of morin derivatives on the uptake of [ 3H]vincristine

The uptake of [ 3H]vincristine by K562 cells and

Fig. 3 represents the effects of 20 mM morin, penta-

Fig. 2. Effects of 20 mM quercetin and its derivatives on the uptake of [ 3H]vincristine by (A) K562 cells and (B) K562/ADM cells. Each bar represents the mean ^ SEM from four experiments. Significant differences from the control were determined by ANOVA followed by Duncan’s test (*; P , 0:05; **; P , 0:01).

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Fig. 3. Effects of 20 mM morin and its derivatives on the uptake of [ 3H]vincristine by (A) K562 cells and (B) K562/ADM cells. Each bar represents the mean ^ SEM from four experiments. Significant differences from the control were determined by ANOVA followed by Duncan’s test (**; P , 0:01).

methoxymorin (M-M), morin pentaethyl ether (M-E), morin pentapropyl ether (M-P), morin pentabutyl ether (M-B) and morin pentaallyl ether (M-A) on the uptake of [ 3H]vincristine by K562 cells and K562/ADM cells, along with that of 20 mM VER and CyA. M-M, M-E, M-P and M-A significantly increased the uptake of [ 3H]vincristine by K562/ ADM cells. Among the above derivatives, the effect of M-E was more potent than typical P-gp inhibitors, VER and CyA (Fig. 3(B)). Although the above four derivatives also significantly increased the uptake of [ 3H]vincristine in K562 cells, the rates of increases were far smaller than that observed in K562/ADM cells (Fig. 3(A)).

4. Discussion We have already reported that flavonoid derivatives such as nobiletin, tangeretin and 3,3 0 ,4 0 ,5,6,7,8-heptamethoxyflavone increase the cellular uptake of vincristine in multidrug-resistant tumor cells by inhibiting the function of P-gp, which actively extrude vincristine from tumor cells [8]. Moreover, we have also reported that pentamethyl, pentaethyl, pentapropyl, pentabutyl and pentaallyl ethers of morin and

quercetin possess antitumor promoter activity. Among these etherified derivatives, pentaallyl ethers exhibited the most potent antitumor promoter activity [9]. If these flavonoid derivatives also possess the inhibitory effects on P-gp activity, they may become candidates of effective agents in cancer chemotherapy. Indeed, Q-A, which has been reported to show remarkable inhibitory effects on tumor promotion [9], inhibited the function of P-gp more potently than the typical P-gp inhibitors, CyA and VER (Fig. 2(B)). Therefore, Q-A was found to become a candidate of multidrug reversing/antitumor promoting agent in cancer chemotherapy. Unlike pentaethyl ethers and pentaallyl ethers of morin and quercetin, pentabutyl ethers of morin and quercetin did not inhibit the function of P-gp. Therefore, the size of alkyloxy (or alkenyloxy) moiety may play a critical role in the inhibition of P-gp. The uptake of [ 3H]vincristine was also increased in parental K562 cells by VER, CyA and flavonoid derivatives. The spectrum of increase in the cellular uptake of [ 3H]vincristine in K562 cells was similar to that in K562/ADM cells. Therefore, the increase in the uptake of [ 3H]vincristine observed in K562 cells may result from the inhibition of P-gp originally expressed in K562 cells. P-gp is not only a cause of

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MDR but also a cause of basal drug resistance of drugnaive tumor cells [5]. Taken together, the present results suggest that these flavonoid derivatives may potentiate the effects of anticancer agents in the treatment of not only multidrug-resistant tumors but also drug-naive tumors. We have reported that the rank order of anticancer promoting activity of flavonoid derivatives is pentaallyl ethers . pentamethyl ethers . pentaethyl ethers . pentapropyl ethers . pentabutyl ethers [9]. This order is different from that of P-gp inhibition in the present study, suggesting that the anticancer promoting activity of flavonoid derivatives may not be attributed to their P-gp inhibitory effects. In this study, we did not investigate the indirect effects of these flavonoid derivatives, such as the effects on the expression level of P-gp. Such indirect effects may be determined by further study with chronic treatment of cells with these compounds. Moreover, toxicological and pharmacokinetic properties of these flavonoid derivatives also remain unclear. They should be determined during the process of in vivo application. In conclusion, Q-E, Q-A and M-E were more potent than VER and CyA in the reversal of MDR. Taking into consideration their antitumor promoter activity, Q-A may become a candidate of effective MDR reversing agent in cancer chemotherapy.

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