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Effects of dietary chemopreventive phytochemicals on P-glycoprotein function
BBRC Biochemical and Biophysical Research Communications 327 (2005) 866–870 www.elsevier.com/locate/ybbrc
Effects of dietary chemopreventive phytochemicals on P-glycoprotein function Tomohiro Nabekura*, Shizu Kamiyama, Shuji Kitagawa Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan Received 9 December 2004 Available online 24 December 2004
Abstract The effects of dietary phytochemicals on P-glycoprotein function were investigated using human multidrug-resistant carcinoma KB-C2 cells and the fluorescent P-glycoprotein substrates daunorubicin and rhodamine 123. The effects of natural chemopreventive compounds, capsaicin found in chilli peppers, curcumin in turmeric, [6]-gingerol in ginger, resveratrol in grapes, sulforaphane in broccoli, 6-methylsulfinyl hexyl isothiocyanate (6-HITC) in Japanese horseradish wasabi, indole-3-carbinol (I3C) in cabbage, and diallyl sulfide and diallyl trisulfide in garlic, were examined. The accumulation of daunorubicin in KB-C2 cells increased in the presence of capsaicin, curcumin, [6]-gingerol, and resveratrol in a concentration-dependent manner. The accumulation of rhodamine 123 in KB-C2 cells was also increased, and the efflux of rhodamine 123 from KB-C2 cells was decreased by these phytochemicals. Sulforaphane, 6-HITC, I3C, and diallyl sulfide and diallyl trisulfide had no effect. These results suggest that dietary phytochemicals, such as capsaicin, curcumin, [6]-gingerol, and resveratrol, have inhibitory effects on P-glycoprotein and potencies to cause drug–food interactions. 2004 Elsevier Inc. All rights reserved. Keywords: P-glycoprotein; Multidrug resistance; Drug–food interaction; Dietary phytochemical; KB cell
Multidrug resistance is a phenomenon of resistance of tumors to chemically unrelated anticancer drugs, and is the one of the most formidable challenges in the field of cancer chemotherapy. Although multiple mechanisms mediate multidrug resistance, the first mediator of multidrug resistance to be characterized at the molecular level was P-glycoprotein (P-gp), a product of MDR1, also referred to as ABCB1, gene [1,2]. P-gp mediates resistance to various classes of chemotherapeutic agents including vinblastine, vincristine, daunorubicin, doxorubicn, colchicine, paclitaxel, and etoposide, by actively extruding the drugs from the cells to lower the intracellular concentrations. The molecular structure of P-gp consists of 12 transmembrane domains that form a drug-binding pore and *
Corresponding author. Fax: +81 25 268 1230. E-mail address: [email protected] (T. Nabekura).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.12.081
two ATP-binding sites belonging to the ATP-binding cassette (ABC) transporter family. Besides anti-cancer agents, various clinically important drugs, including digoxin, verapamil, cyclosporin A, tacrolimus, progesterone, and saquinavir, are substrates of P-gp. P-gp is also expressed in normal tissues. It is abundant in the apical membranes of many pharmacologically important epithelial barriers, such as the intestinal epithelium, renal proximal tubular epithelium, and the blood–brain barrier [1,2]. Therefore, it is considered that P-gp plays very important roles in the absorption, distribution, and elimination of many commonly used drugs, and thus determines the efficacy and toxicity of drugs. Many molecular and cellular studies have revealed the ability of dietary phytochemicals in vegetables and fruits to reduce the risk of cancer. Recently, dietary supplements and/or herbal remedies are widely used by older adults, often concomitantly with prescribed
T. Nabekura et al. / Biochemical and Biophysical Research Communications 327 (2005) 866–870
medications. However, less attention has been paid to the interactions between drugs and food, and the effects of food components on the function of drug transporters, such as P-gp, have not fully investigated. In this study, we investigated the effects of dietary phytochemicals that are reported to possess cancer preventive potential on the function of P-gp using P-gp-mediated multidrug-resistant human carcinoma KB-C2 cells.
Materials and methods Materials. DulbeccoÕs modified EagleÕs medium (D-MEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Colchicine, daunorubicin, rhodamine 123, vinblastine, capsaicin, and [6]-gingerol were from Wako Pure Chemical Industries (Osaka, Japan). Curcumin, resveratrol, and indole-3-carbinol (I3C) were from Sigma Chemical (St. Louis, MO). Sulforaphane, 6-methylsulfinyl hexyl isothyocyanate (6-HITC), diallyl sulfide, and diallyl trisulfide were from LKT Laboratories (St. Paul, MN). Fig. 1 shows the chemical structures and sources of several dietary phytochemicals. All other chemicals used were of the highest purity available. KB-C2 cells were kindly provided by Prof. Shin-ichi Akiyama of Kagoshima University (Kagoshima, Japan). Cell culture. KB-C2 cells were cultured in D-MEM culture medium supplemented with 10% FBS and 2 lg/mL colchicine. Cells were incubated at 37 C in a humidified atmosphere with 5% CO2–95% air. Measurement of the cellular accumulation of daunorubicin. The accumulation of daunorubicin, a fluorescence substrate of P-gp, was measured and the effects of dietary phytochemicals were determined as described previously [3]. Briefly, KB-C2 cells, plated at 2.5 · 105 cells/ 35-mm dish, were incubated with 50 lM daunorubicin in the absence or presence of phytochemicals for 2 h in a CO2 incubator at 37 C.
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After incubation, the medium was removed by aspiration and the cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed with 1% sodium dodecyl sulfate (SDS) in PBS. Fluorescence intensity was measured by a spectrofluorometer (F-4010, Hitachi, Tokyo, Japan). The excitation and emission wavelengths were 502 and 588 nm, respectively. Accumulation ratios were calculated using the accumulation of daunorubicin in cells incubated without phytochemicals as a control. Measurement of cellular accumulation of rhodamine 123. The accumulation of rhodamine 123, a fluorescent substrate of P-gp, in KB-C2 cells was measured using a FACS flowcytometer (FACScalibur, BD Biosciences, San Jose, CA) equipped with an ultraviolet argon laser (excitation at 488 nm and emission at 530 ± 15 nm), as described previously [3]. Cells were incubated with 20 lM rhodamine 123 in the absence or presence of 50 lM dietary phytochemicals for 2 h at 37 C. After incubation, the cells were washed with ice-cold PBS, and the fluorescence intensity of rhodamine 123 in individual cells was measured immediately by a flowcytometer. For the efflux experiment, cells were incubated with 20 lM rhodamine 123 in the absence of phytochemicals for 2 h at 37 C. After 2 h incubation, the cells were washed with ice-cold PBS, the medium was replaced with rhodamine 123-free medium containing 50 lM phytochemicals and further incubated for 30 min at 37 C. After 30 min incubation, the cells were washed with ice-cold PBS and rhodamine 123 retained in the cells was measured by flowcytometry. Determination of resistance to vinblastine cytotoxicity. The resistance of KB-C2 cells to vinblastine cytotoxicity was determined by a calorimetric assay using a new water-soluble tetrazolium salt, 2-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt (WST-8) (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan), performed in 96-well plates [4,5]. 5 · 103 KB-C2 cells in 180 ll of culture medium in the absence or presence of phytochemicals were inoculated into each well. After 24 h incubation in a CO2 incubator at 37 C, 20 ll of various concentrations of vinblastine was added and the plates were incubated for 3 days. Thereafter, the medium was removed by aspiration, and 90 ll of fresh culture medium and 10 ll of Cell Counting Kit-8 solution (5 mM WST-8) were added to each well. The plates were incubated for a further 4 h, and the absorbance at 450 nm was measured using a microplate reader (model 550, Bio-Rad Laboratories, Hercules, CA). Statistical analysis. Data are expressed as means ± standard error of the mean (SE). Statistical differences were determined by one-way analysis of variance (ANOVA) followed by FisherÕs test. P values less than 0.05 were considered significant.
Results Effects of phytochemicals on daunorubicin accumulation
Fig. 1. Chemical structures and sources of dietary phytochemicals.
Fig. 2 shows the accumulation ratio of daunorubicin in KB-C2 cells in the presence of 50 lM dietary phytochemicals. Capsaicin, curcumin, [6]-gingerol, and resveratrol increased the cellular accumulation of daunorubicin, which indicates that these phytochemicals inhibit the Pgp-mediated efflux of daunorubicin. In contrast, sulforaphane, 6-HITC, I3C, diallyl sulfide, and diallyl trisulfide had no effects on the cellular accumulation of daunorubicin. Higher concentrations (100 lM) of sulforaphane, 6HITC, I3C, diallyl sulfide, and diallyl trisulfide also had no effects on intracellular accumulation (sulforaphane, 1.004 ± 0.039; 6-HITC, 1.029 ± 0.061; I3C, 0.974 ± 0.047; diallyl sulfide, 0.771 ± 0.037; and diallyl trisulfide,
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Fig. 2. Effects of 50 lM dietary phytochemicals on daunorubicin accumulation in KB-C2 cells. 6-HITC, 6-methylsulfinyl hexyl isothyocyanate; I3C, indole-3-carbinol. Each column represents the mean + SE of six experiments. *P < 0.05, significantly different from control.
Fig. 3. Effects of capsaicin, curcumin, [6]-gingerol, and resveratrol on daunorubicin accumulation in KB-C2 cells. Capsaicin (s), curcumin (d), [6]-gingerol (n), and resveratrol (m). Mean ± SE of six experiments. *P < 0.05, significantly different from control.
0.714 ± 0.044, accumulation ratio, mean ± SE of six experiments). Fig. 3 shows the accumulation ratio of daunorubicin in KB-C2 cells in the presence of various concentrations of phytochemicals. Capsaicin, curcumin, [6]-gingerol, and resveratrol increased the cellular accumulation of daunorubicin in a concentration-dependent manner. Effects of phytochemicals on the accumulation and efflux of rhodamine 123 Fig. 4 shows the accumulation of rhodamine 123 in individual cells as analyzed by FACS flowcytometry. Capsaicin, curcumin, and [6]-gingerol increased the cellular accumulation of rhodamine 123 in KB-C2 cells (capsaicin, 4.004 ± 0.324*; curcumin, 2.449 ± 0.467*; [6]-gingerol, 2.243 ± 0.243*; and resveratrol, 1.361 ± 0.170, accumulation ratio, mean ± SE of five experiments, *significantly different from control, P < 0.05).
Fig. 4. Accumulation of rhodamine 123 in KB-C2 cells. Accumulation of rhodamine 123 in individual KB-C2 cells in the absence or presence of 50 lM phytochemicals was analyzed by FACS flowcytometry.
Fig. 5. Efflux of rhodamine 123 from KB-C2 cells. Efflux of rhodamine 123 from KB-C2 cells in the absence or presence of 50 lM phytochemicals was analyzed by FACS flowcytometry.
Fig. 5 shows the efflux of rhodamine 123 from KB-C2 cells and the effects of phytochemicals. After 2 h incubation with rhodamine 123, KB-C2 cells showed high fluorescence intensity (Fig. 5, thin line). Further incubation with rhodamine 123-free medium decreased the fluorescence intensity of cells due to a decrease in rhodamine 123 in cells caused by P-gp-mediated efflux (Fig. 5, thick line). In the presence of capsaicin, curcumin, [6]-gingerol, and resveratrol in rhodamine 123-free medium, fluorescence intensities were higher than that of medium without phytochemicals. Effects of phytochemicals on vinblastine cytotoxicity Fig. 6 shows cell growth inhibition curves of vinblastine in the absence and presence of capsaicin and [6]gingerol. In the presence of capsaicin and [6]-gingerol, KB-C2 cells were more susceptible to the cytotoxicity of vinblastine, a P-gp substrate, as compared with
T. Nabekura et al. / Biochemical and Biophysical Research Communications 327 (2005) 866–870
Fig. 6. Effects of capsaicin and [6]-gingerol on vinblastine cytotoxicity. Control (s), 50 lM capsaicin (d), and 50 lM [6]-gingerol (m). Mean ± SE of six experiments. *P < 0.05, significantly different from control.
vinblastine alone, indicating the inhibition of P-gpmediated vinblastine efflux from cells and an increase in intracellular vinblastine concentrations in response to capsaicin and [6]-gingerol. At a concentration of 50 lM, curcumin itself showed cytotoxic effects on KB-C2 cells and no effects on vinblastine sensitivity were detected. We also could not detect effects on vinblastine cytotoxicity of 50 lM resveratrol, probably due to its low affinity for P-gp.
Discussion Over the last few years, the effects of natural products in cancer treatment or prevention have been extensively studied. Tea (Camellia sinensis) consumption has been shown to inhibit tumor formation at different organ sites including the skin, oral cavity, esophagus, stomach, intestine, lung, liver, pancreas, mammary gland, urinary bladder, and prostate [6,7]. ( )-Epigallocatechin gallate (EGCG), a major water-extractable constituent of tea, has been presumed to be the active compound for the cancer preventive effects. In addition to the anti-carcinogenic effects of EGCG, we previously revealed the inhibitory effects of EGCG and other tea catechins on P-gp function using human carcinoma KB-C2 cells and fluorescent P-gp substrates daunorubicin and rhodamine 123 [3]. KB-C2 is a multidrug-resistant human epidermal carcinoma cell line that overexpresses P-gp, a drug efflux transporter. Daunorubicin and rhodamine 123 have often been used in studies of P-gp-mediated transport. Daunorubicin is also reported to be a substrate of multidrug resistance protein 1 (MRP1). However, MRP1 is scarcely found in KB-C2 cells, and its involvement in daunorubicin accumulation in KB-C2 cell is negligible. In the previous study, we showed that tea catechins increased the cellular accumulation of daunorubicin and rhodamine 123 in the order of ( )-epigallocatechin < ( )-epicatechin gallate < EGCG [3]. In the present study, we further examined the effects of dietary phytochemicals commonly found in consumed foods on the function of P-gp.
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Capsaicin, a pungent component of hot chilli pepper (Capsicum annuum), has been suspected to act as a carcinogen or a co-carcinogen in experimental animals because of its irritant properties, but other studies indicate that the compound has chemopreventive effects [6,7]. Capsaicin inhibits the constitutive and induced activation of nuclear factor-kappa B (NF-jB) in human malignant melanoma cells, leading to an inhibition of melanoma cell proliferation. Capsaicin also induces apoptosis in human mammary and Jurkat cells. Curcumin is a yellow pigment that is present in turmeric (Curcuma longa) and related species. Curcumin has strong anti-oxidant and anti-inflammatory properties, and is reported to inhibit chemically induced skin, stomach, and colon carcinogenesis in post-initiation stages in animals. [6]-Gingerol is a phenolic substance responsible for the spicy taste of ginger (Zingiber officinale). It is reported that [6]-gingerol inhibits tumor promotion in mouse skin and epidermal growth factor-induced cell transformation. Resveratrol is a phytoalexin present in grapes (Vitis vinifera) and a key anti-oxidant ingredient of red wine. Resveratrol induces apoptosis, reduces the constitutive activation of NF-jB, and suppresses the proliferation of human leukemia, pancreas, and breast cancer cells. In addition to the phytochemicals descried above, organic isothiocyanates, sulforaphane found in broccoli, 6-HITC in Japanese horseradish wasabi, and I3C in cabbage, and the organosulfur compounds diallyl sulfide and diallyl trisulfide present in garlic have also been reported to have chemopreventive effects and to suppress tumor promotion or progression in experimental animals [6,7]. Sulforaphane, 6-HITC, I3C, diallyl sulfide, and diallyl trisulfide had no effects on the cellular accumulation of daunorubicin in KB-C2 cells (Fig. 2). On the other hand, capsaicin, curcumin, [6]-gingerol, and resveratrol increased the accumulation of daunorubicin in a concentration-dependent manner (Figs. 2 and 3). Since these phytochemicals inhibit the efflux of P-gp substrates, the elevation of substrate accumulation seems to be induced by the inhibition of the efflux transporter. The cellular accumulation of rhodamine 123 was also increased in the presence of these compounds (Fig. 4). The efflux of rhodamine 123 from KB-C2 cells was decreased by capsaicin, curcumin, [6]-gingerol, and resveratrol (Fig. 5). Capsaicin and [6]-gingerol increased the sensitivity to vinblastine cytotoxicity of KB-C2 cells (Fig. 6). This demonstrates that capsaicin and [6]-gingerol can partially reverse multidrug resistance in cells that express P-gp. Taken together, the present results indicate that capsaicin, curcumin, [6]-gingerol, and resveratrol have inhibitory effects on P-gp function, and the possibility of causing food–drug interactions by modulating the activity of P-gp. It is now established that nutrients and phytochemicals can have pronounced impact on drug absorption,
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disposition, and elimination [8,9]. Grapefruit juice and the mild antidepressant St. JohnÕs wort (Hypericum perforatum) are a well-known example of a food–drug interaction. Some dietary phytochemicals, such as the bioflavonoid quercetin, morin, and EGCG, have been evaluated for their potential P-gp inhibitory effects [3,10–12]. Recently, Anuchapreeda et al. [13] reported that curcumin has an inhibitory effect on P-gp function. Foster et al. [14] examined the effects of garlic products on P-gp function, but only slight effects could be observed. These results are comparable to ours that curcumin has an inhibitory effect on P-gp, but that the garlic constituents diallyl sulfide and diallyl trisulfide do not. It is interesting that Anuchapreeda et al. [13] also examined the effects of chronic exposure of curcumin to cells and the expression of P-gp. They showed that curcumin at concentrations of 1 to 10 lM for up to 72 h decreased P-gp expression and MDR1 mRNA levels. To clarify the effects of dietary phytochemicals on drug disposition, it is important to investigate the effects of long-term exposure and modulation of P-gp expression. Therefore, we are now in the process of investigating the effects of capsaicin and [6]-gingerol on the expression of P-gp and MDR1 mRNA in KB-C2 and human colon carcinoma Caco-2 and LS180 cell lines. In conclusion, we used P-gp overexpressed multidrug-resistant human carcinoma KB-C2 cells and fluorescent P-gp substrates daunorubicin and rhodamine 123 to investigate the effects of dietary phytochemicals on P-gp function. Several naturally chemopreventive compounds in vegetables and fruits, such as capsaicin, curcumin, [6]-gingerol, and resveratrol, have inhibitory effects on P-gp and potencies to cause food–drug interactions. Capsaicin, curcumin, [6]-gingerol, and resveratrol can be considered as promising lead compounds for the design of more efficacious and low toxicity compounds to reverse multidrug resistance in cancer. These phytochemicals could also be useful for further studies of positive uses of food–drug interactions as a strategy to enhance the bioavailability and/or prolonged effects of P-gp substrate drugs.
Acknowledgments This work was supported by a Grant-in-Aid for the Encouragement of Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Sasakawa Scientific Research Grant from the Japan Science Society, and The Danone Institute of Japan, The 2004 DIJ Research Grant (T.N.), and a
grant from the Promotion and Mutual Aid Corporation for Private Schools in Japan.
References [1] S.V. Ambudkar, S. Dey, C.A. Hrycyna, M. Ramachandra, I. Pastan, M.M. Gottesman, Biochemical, cellular, and pharmacological aspects of the multidrug transporter, Annu. Rev. Pharmacol. Toxicol. 39 (1999) 361–398. [2] W.W. Johnson, ABC transporter proteins and cellular drug resistance: P-glycoprotein and analogous transporters, in: D.R. Lu, S. Øie (Eds.), Cellular Drug Delivery: Principles and Practice, Humana Press, Totowa, NJ, 2004, pp. 129–162. [3] S. Kitagawa, T. Nabekura, S. Kamiyama, Inhibition of Pglycoprotein function by tea catechins in KB-C2 cells, J. Pharm. Pharmacol. 56 (2004) 1001–1005. [4] H. Tominaga, M. Ishiyama, F. Ohseto, K. Sasamoto, T. Hamamoto, K. Suzuki, M. Watanabe, A water-soluble tetrazolium salt useful for calorimetric cell viability assay, Anal. Commun. 36 (1999) 47–50. [5] H. Okumura, Z.-S. Chen, M. Sakou, T. Sumizawa, T. Furukawa, M. Komatsu, R. Ikeda, H. Suzuki, K. Hirota, T. Aikou, S.-I. Akiyama, Reversal of P-glycoprotein and multidrug-resistance protein-mediated drug resistance in KB cells by 5-O-benzoylated taxinine K, Mol. Pharmacol. 58 (2000) 1563–1569. [6] Y.-J. Surh, Cancer chemoprevention with dietary phytochemicals, Nat. Rev. Cancer 3 (2003) 768–780. [7] H. Tsuda, Y. Ohshima, H. Nomoto, K.-I. Fujita, E. Matsuda, M. Iigo, N. Takasuka, M.A. Moore, Cancer prevention by natural compounds, Drug Metab. Pharmacokinet. 19 (2004) 245– 263. [8] S.-M. Huang, S.D. Hall, P. Watkins, L.A. Love, C. SerabjitSingh, J.M. Betz, F.A. Hoffman, P. Honig, P.M. Coates, J. Bull, S.T. Chen, G.L. Kearns, M.D. Murray, Drug interactions with herbal products and grapefruit juice: a conference report, Clin. Pharmacol. Ther. 75 (2004) 1–12. [9] A. Dahan, H. Altman, Food–drug interaction: grapefruit juice augments drug bioavailability—mechanism, extent and relevance, Eur. J. Clin. Nutr. 58 (2004) 1–9. [10] G. Conseil, H. Baubichon-Cortay, G. Dayan, J.-M. Jault, D. Barron, A. DiPietro, Flavonoids: a class of modulators with bifunctional interactions at vicinal ATP- and steroid-binding sites on mouse P-glycoprotein, Proc. Natl. Acad. Sci. USA 95 (1998) 9831–9836. [11] S. Zhang, M.E. Morris, Effects of the flavonoids biochanin A, morin, phloretin, and silymarin on P-glycoprotein-mediated transport, J. Pharmacol. Exp. Ther. 304 (2003) 1258–1267. [12] J. Jodoin, M. Demeule, R. Be´liveau, Inhibition of the multidrug resistance P-glycoprotein activity by green tea polyphenols, Biochim. Biophys. Acta 1542 (2002) 149–159. [13] S. Anuchapreeda, P. Leechanachai, M.M. Smith, S.V. Ambudkar, P.-N. Limtrakul, Modulation of P-glycoprotein expression and function by curcumin in multidrug-resistant human KB cells, Biochem. Pharmacol. 64 (2002) 573–582. [14] B.C. Foster, M.S. Foster, S. Vandenhoek, A. Krantis, J.W. Budzinski, J.T. Arnason, K.D. Gallicano, S. Choudri, B.C. Foster, An in vitro evaluation of human cytochrome P450 3A4 and P-glycoprotein inhibition by garlic, J. Pharm. Pharm. Sci. 4 (2001) 176–184.
Effects of dietary chemopreventive phytochemicals on P-glycoprotein function Tomohiro Nabekura*, Shizu Kamiyama, Shuji Kitagawa Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan Received 9 December 2004 Available online 24 December 2004
Abstract The effects of dietary phytochemicals on P-glycoprotein function were investigated using human multidrug-resistant carcinoma KB-C2 cells and the fluorescent P-glycoprotein substrates daunorubicin and rhodamine 123. The effects of natural chemopreventive compounds, capsaicin found in chilli peppers, curcumin in turmeric, [6]-gingerol in ginger, resveratrol in grapes, sulforaphane in broccoli, 6-methylsulfinyl hexyl isothiocyanate (6-HITC) in Japanese horseradish wasabi, indole-3-carbinol (I3C) in cabbage, and diallyl sulfide and diallyl trisulfide in garlic, were examined. The accumulation of daunorubicin in KB-C2 cells increased in the presence of capsaicin, curcumin, [6]-gingerol, and resveratrol in a concentration-dependent manner. The accumulation of rhodamine 123 in KB-C2 cells was also increased, and the efflux of rhodamine 123 from KB-C2 cells was decreased by these phytochemicals. Sulforaphane, 6-HITC, I3C, and diallyl sulfide and diallyl trisulfide had no effect. These results suggest that dietary phytochemicals, such as capsaicin, curcumin, [6]-gingerol, and resveratrol, have inhibitory effects on P-glycoprotein and potencies to cause drug–food interactions. 2004 Elsevier Inc. All rights reserved. Keywords: P-glycoprotein; Multidrug resistance; Drug–food interaction; Dietary phytochemical; KB cell
Multidrug resistance is a phenomenon of resistance of tumors to chemically unrelated anticancer drugs, and is the one of the most formidable challenges in the field of cancer chemotherapy. Although multiple mechanisms mediate multidrug resistance, the first mediator of multidrug resistance to be characterized at the molecular level was P-glycoprotein (P-gp), a product of MDR1, also referred to as ABCB1, gene [1,2]. P-gp mediates resistance to various classes of chemotherapeutic agents including vinblastine, vincristine, daunorubicin, doxorubicn, colchicine, paclitaxel, and etoposide, by actively extruding the drugs from the cells to lower the intracellular concentrations. The molecular structure of P-gp consists of 12 transmembrane domains that form a drug-binding pore and *
Corresponding author. Fax: +81 25 268 1230. E-mail address: [email protected] (T. Nabekura).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.12.081
two ATP-binding sites belonging to the ATP-binding cassette (ABC) transporter family. Besides anti-cancer agents, various clinically important drugs, including digoxin, verapamil, cyclosporin A, tacrolimus, progesterone, and saquinavir, are substrates of P-gp. P-gp is also expressed in normal tissues. It is abundant in the apical membranes of many pharmacologically important epithelial barriers, such as the intestinal epithelium, renal proximal tubular epithelium, and the blood–brain barrier [1,2]. Therefore, it is considered that P-gp plays very important roles in the absorption, distribution, and elimination of many commonly used drugs, and thus determines the efficacy and toxicity of drugs. Many molecular and cellular studies have revealed the ability of dietary phytochemicals in vegetables and fruits to reduce the risk of cancer. Recently, dietary supplements and/or herbal remedies are widely used by older adults, often concomitantly with prescribed
T. Nabekura et al. / Biochemical and Biophysical Research Communications 327 (2005) 866–870
medications. However, less attention has been paid to the interactions between drugs and food, and the effects of food components on the function of drug transporters, such as P-gp, have not fully investigated. In this study, we investigated the effects of dietary phytochemicals that are reported to possess cancer preventive potential on the function of P-gp using P-gp-mediated multidrug-resistant human carcinoma KB-C2 cells.
Materials and methods Materials. DulbeccoÕs modified EagleÕs medium (D-MEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Colchicine, daunorubicin, rhodamine 123, vinblastine, capsaicin, and [6]-gingerol were from Wako Pure Chemical Industries (Osaka, Japan). Curcumin, resveratrol, and indole-3-carbinol (I3C) were from Sigma Chemical (St. Louis, MO). Sulforaphane, 6-methylsulfinyl hexyl isothyocyanate (6-HITC), diallyl sulfide, and diallyl trisulfide were from LKT Laboratories (St. Paul, MN). Fig. 1 shows the chemical structures and sources of several dietary phytochemicals. All other chemicals used were of the highest purity available. KB-C2 cells were kindly provided by Prof. Shin-ichi Akiyama of Kagoshima University (Kagoshima, Japan). Cell culture. KB-C2 cells were cultured in D-MEM culture medium supplemented with 10% FBS and 2 lg/mL colchicine. Cells were incubated at 37 C in a humidified atmosphere with 5% CO2–95% air. Measurement of the cellular accumulation of daunorubicin. The accumulation of daunorubicin, a fluorescence substrate of P-gp, was measured and the effects of dietary phytochemicals were determined as described previously [3]. Briefly, KB-C2 cells, plated at 2.5 · 105 cells/ 35-mm dish, were incubated with 50 lM daunorubicin in the absence or presence of phytochemicals for 2 h in a CO2 incubator at 37 C.
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After incubation, the medium was removed by aspiration and the cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed with 1% sodium dodecyl sulfate (SDS) in PBS. Fluorescence intensity was measured by a spectrofluorometer (F-4010, Hitachi, Tokyo, Japan). The excitation and emission wavelengths were 502 and 588 nm, respectively. Accumulation ratios were calculated using the accumulation of daunorubicin in cells incubated without phytochemicals as a control. Measurement of cellular accumulation of rhodamine 123. The accumulation of rhodamine 123, a fluorescent substrate of P-gp, in KB-C2 cells was measured using a FACS flowcytometer (FACScalibur, BD Biosciences, San Jose, CA) equipped with an ultraviolet argon laser (excitation at 488 nm and emission at 530 ± 15 nm), as described previously [3]. Cells were incubated with 20 lM rhodamine 123 in the absence or presence of 50 lM dietary phytochemicals for 2 h at 37 C. After incubation, the cells were washed with ice-cold PBS, and the fluorescence intensity of rhodamine 123 in individual cells was measured immediately by a flowcytometer. For the efflux experiment, cells were incubated with 20 lM rhodamine 123 in the absence of phytochemicals for 2 h at 37 C. After 2 h incubation, the cells were washed with ice-cold PBS, the medium was replaced with rhodamine 123-free medium containing 50 lM phytochemicals and further incubated for 30 min at 37 C. After 30 min incubation, the cells were washed with ice-cold PBS and rhodamine 123 retained in the cells was measured by flowcytometry. Determination of resistance to vinblastine cytotoxicity. The resistance of KB-C2 cells to vinblastine cytotoxicity was determined by a calorimetric assay using a new water-soluble tetrazolium salt, 2-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt (WST-8) (Cell Counting Kit-8, Dojindo Laboratories, Kumamoto, Japan), performed in 96-well plates [4,5]. 5 · 103 KB-C2 cells in 180 ll of culture medium in the absence or presence of phytochemicals were inoculated into each well. After 24 h incubation in a CO2 incubator at 37 C, 20 ll of various concentrations of vinblastine was added and the plates were incubated for 3 days. Thereafter, the medium was removed by aspiration, and 90 ll of fresh culture medium and 10 ll of Cell Counting Kit-8 solution (5 mM WST-8) were added to each well. The plates were incubated for a further 4 h, and the absorbance at 450 nm was measured using a microplate reader (model 550, Bio-Rad Laboratories, Hercules, CA). Statistical analysis. Data are expressed as means ± standard error of the mean (SE). Statistical differences were determined by one-way analysis of variance (ANOVA) followed by FisherÕs test. P values less than 0.05 were considered significant.
Results Effects of phytochemicals on daunorubicin accumulation
Fig. 1. Chemical structures and sources of dietary phytochemicals.
Fig. 2 shows the accumulation ratio of daunorubicin in KB-C2 cells in the presence of 50 lM dietary phytochemicals. Capsaicin, curcumin, [6]-gingerol, and resveratrol increased the cellular accumulation of daunorubicin, which indicates that these phytochemicals inhibit the Pgp-mediated efflux of daunorubicin. In contrast, sulforaphane, 6-HITC, I3C, diallyl sulfide, and diallyl trisulfide had no effects on the cellular accumulation of daunorubicin. Higher concentrations (100 lM) of sulforaphane, 6HITC, I3C, diallyl sulfide, and diallyl trisulfide also had no effects on intracellular accumulation (sulforaphane, 1.004 ± 0.039; 6-HITC, 1.029 ± 0.061; I3C, 0.974 ± 0.047; diallyl sulfide, 0.771 ± 0.037; and diallyl trisulfide,
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Fig. 2. Effects of 50 lM dietary phytochemicals on daunorubicin accumulation in KB-C2 cells. 6-HITC, 6-methylsulfinyl hexyl isothyocyanate; I3C, indole-3-carbinol. Each column represents the mean + SE of six experiments. *P < 0.05, significantly different from control.
Fig. 3. Effects of capsaicin, curcumin, [6]-gingerol, and resveratrol on daunorubicin accumulation in KB-C2 cells. Capsaicin (s), curcumin (d), [6]-gingerol (n), and resveratrol (m). Mean ± SE of six experiments. *P < 0.05, significantly different from control.
0.714 ± 0.044, accumulation ratio, mean ± SE of six experiments). Fig. 3 shows the accumulation ratio of daunorubicin in KB-C2 cells in the presence of various concentrations of phytochemicals. Capsaicin, curcumin, [6]-gingerol, and resveratrol increased the cellular accumulation of daunorubicin in a concentration-dependent manner. Effects of phytochemicals on the accumulation and efflux of rhodamine 123 Fig. 4 shows the accumulation of rhodamine 123 in individual cells as analyzed by FACS flowcytometry. Capsaicin, curcumin, and [6]-gingerol increased the cellular accumulation of rhodamine 123 in KB-C2 cells (capsaicin, 4.004 ± 0.324*; curcumin, 2.449 ± 0.467*; [6]-gingerol, 2.243 ± 0.243*; and resveratrol, 1.361 ± 0.170, accumulation ratio, mean ± SE of five experiments, *significantly different from control, P < 0.05).
Fig. 4. Accumulation of rhodamine 123 in KB-C2 cells. Accumulation of rhodamine 123 in individual KB-C2 cells in the absence or presence of 50 lM phytochemicals was analyzed by FACS flowcytometry.
Fig. 5. Efflux of rhodamine 123 from KB-C2 cells. Efflux of rhodamine 123 from KB-C2 cells in the absence or presence of 50 lM phytochemicals was analyzed by FACS flowcytometry.
Fig. 5 shows the efflux of rhodamine 123 from KB-C2 cells and the effects of phytochemicals. After 2 h incubation with rhodamine 123, KB-C2 cells showed high fluorescence intensity (Fig. 5, thin line). Further incubation with rhodamine 123-free medium decreased the fluorescence intensity of cells due to a decrease in rhodamine 123 in cells caused by P-gp-mediated efflux (Fig. 5, thick line). In the presence of capsaicin, curcumin, [6]-gingerol, and resveratrol in rhodamine 123-free medium, fluorescence intensities were higher than that of medium without phytochemicals. Effects of phytochemicals on vinblastine cytotoxicity Fig. 6 shows cell growth inhibition curves of vinblastine in the absence and presence of capsaicin and [6]gingerol. In the presence of capsaicin and [6]-gingerol, KB-C2 cells were more susceptible to the cytotoxicity of vinblastine, a P-gp substrate, as compared with
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Fig. 6. Effects of capsaicin and [6]-gingerol on vinblastine cytotoxicity. Control (s), 50 lM capsaicin (d), and 50 lM [6]-gingerol (m). Mean ± SE of six experiments. *P < 0.05, significantly different from control.
vinblastine alone, indicating the inhibition of P-gpmediated vinblastine efflux from cells and an increase in intracellular vinblastine concentrations in response to capsaicin and [6]-gingerol. At a concentration of 50 lM, curcumin itself showed cytotoxic effects on KB-C2 cells and no effects on vinblastine sensitivity were detected. We also could not detect effects on vinblastine cytotoxicity of 50 lM resveratrol, probably due to its low affinity for P-gp.
Discussion Over the last few years, the effects of natural products in cancer treatment or prevention have been extensively studied. Tea (Camellia sinensis) consumption has been shown to inhibit tumor formation at different organ sites including the skin, oral cavity, esophagus, stomach, intestine, lung, liver, pancreas, mammary gland, urinary bladder, and prostate [6,7]. ( )-Epigallocatechin gallate (EGCG), a major water-extractable constituent of tea, has been presumed to be the active compound for the cancer preventive effects. In addition to the anti-carcinogenic effects of EGCG, we previously revealed the inhibitory effects of EGCG and other tea catechins on P-gp function using human carcinoma KB-C2 cells and fluorescent P-gp substrates daunorubicin and rhodamine 123 [3]. KB-C2 is a multidrug-resistant human epidermal carcinoma cell line that overexpresses P-gp, a drug efflux transporter. Daunorubicin and rhodamine 123 have often been used in studies of P-gp-mediated transport. Daunorubicin is also reported to be a substrate of multidrug resistance protein 1 (MRP1). However, MRP1 is scarcely found in KB-C2 cells, and its involvement in daunorubicin accumulation in KB-C2 cell is negligible. In the previous study, we showed that tea catechins increased the cellular accumulation of daunorubicin and rhodamine 123 in the order of ( )-epigallocatechin < ( )-epicatechin gallate < EGCG [3]. In the present study, we further examined the effects of dietary phytochemicals commonly found in consumed foods on the function of P-gp.
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Capsaicin, a pungent component of hot chilli pepper (Capsicum annuum), has been suspected to act as a carcinogen or a co-carcinogen in experimental animals because of its irritant properties, but other studies indicate that the compound has chemopreventive effects [6,7]. Capsaicin inhibits the constitutive and induced activation of nuclear factor-kappa B (NF-jB) in human malignant melanoma cells, leading to an inhibition of melanoma cell proliferation. Capsaicin also induces apoptosis in human mammary and Jurkat cells. Curcumin is a yellow pigment that is present in turmeric (Curcuma longa) and related species. Curcumin has strong anti-oxidant and anti-inflammatory properties, and is reported to inhibit chemically induced skin, stomach, and colon carcinogenesis in post-initiation stages in animals. [6]-Gingerol is a phenolic substance responsible for the spicy taste of ginger (Zingiber officinale). It is reported that [6]-gingerol inhibits tumor promotion in mouse skin and epidermal growth factor-induced cell transformation. Resveratrol is a phytoalexin present in grapes (Vitis vinifera) and a key anti-oxidant ingredient of red wine. Resveratrol induces apoptosis, reduces the constitutive activation of NF-jB, and suppresses the proliferation of human leukemia, pancreas, and breast cancer cells. In addition to the phytochemicals descried above, organic isothiocyanates, sulforaphane found in broccoli, 6-HITC in Japanese horseradish wasabi, and I3C in cabbage, and the organosulfur compounds diallyl sulfide and diallyl trisulfide present in garlic have also been reported to have chemopreventive effects and to suppress tumor promotion or progression in experimental animals [6,7]. Sulforaphane, 6-HITC, I3C, diallyl sulfide, and diallyl trisulfide had no effects on the cellular accumulation of daunorubicin in KB-C2 cells (Fig. 2). On the other hand, capsaicin, curcumin, [6]-gingerol, and resveratrol increased the accumulation of daunorubicin in a concentration-dependent manner (Figs. 2 and 3). Since these phytochemicals inhibit the efflux of P-gp substrates, the elevation of substrate accumulation seems to be induced by the inhibition of the efflux transporter. The cellular accumulation of rhodamine 123 was also increased in the presence of these compounds (Fig. 4). The efflux of rhodamine 123 from KB-C2 cells was decreased by capsaicin, curcumin, [6]-gingerol, and resveratrol (Fig. 5). Capsaicin and [6]-gingerol increased the sensitivity to vinblastine cytotoxicity of KB-C2 cells (Fig. 6). This demonstrates that capsaicin and [6]-gingerol can partially reverse multidrug resistance in cells that express P-gp. Taken together, the present results indicate that capsaicin, curcumin, [6]-gingerol, and resveratrol have inhibitory effects on P-gp function, and the possibility of causing food–drug interactions by modulating the activity of P-gp. It is now established that nutrients and phytochemicals can have pronounced impact on drug absorption,
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disposition, and elimination [8,9]. Grapefruit juice and the mild antidepressant St. JohnÕs wort (Hypericum perforatum) are a well-known example of a food–drug interaction. Some dietary phytochemicals, such as the bioflavonoid quercetin, morin, and EGCG, have been evaluated for their potential P-gp inhibitory effects [3,10–12]. Recently, Anuchapreeda et al. [13] reported that curcumin has an inhibitory effect on P-gp function. Foster et al. [14] examined the effects of garlic products on P-gp function, but only slight effects could be observed. These results are comparable to ours that curcumin has an inhibitory effect on P-gp, but that the garlic constituents diallyl sulfide and diallyl trisulfide do not. It is interesting that Anuchapreeda et al. [13] also examined the effects of chronic exposure of curcumin to cells and the expression of P-gp. They showed that curcumin at concentrations of 1 to 10 lM for up to 72 h decreased P-gp expression and MDR1 mRNA levels. To clarify the effects of dietary phytochemicals on drug disposition, it is important to investigate the effects of long-term exposure and modulation of P-gp expression. Therefore, we are now in the process of investigating the effects of capsaicin and [6]-gingerol on the expression of P-gp and MDR1 mRNA in KB-C2 and human colon carcinoma Caco-2 and LS180 cell lines. In conclusion, we used P-gp overexpressed multidrug-resistant human carcinoma KB-C2 cells and fluorescent P-gp substrates daunorubicin and rhodamine 123 to investigate the effects of dietary phytochemicals on P-gp function. Several naturally chemopreventive compounds in vegetables and fruits, such as capsaicin, curcumin, [6]-gingerol, and resveratrol, have inhibitory effects on P-gp and potencies to cause food–drug interactions. Capsaicin, curcumin, [6]-gingerol, and resveratrol can be considered as promising lead compounds for the design of more efficacious and low toxicity compounds to reverse multidrug resistance in cancer. These phytochemicals could also be useful for further studies of positive uses of food–drug interactions as a strategy to enhance the bioavailability and/or prolonged effects of P-gp substrate drugs.
Acknowledgments This work was supported by a Grant-in-Aid for the Encouragement of Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Sasakawa Scientific Research Grant from the Japan Science Society, and The Danone Institute of Japan, The 2004 DIJ Research Grant (T.N.), and a
grant from the Promotion and Mutual Aid Corporation for Private Schools in Japan.
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