Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function

G Model

ARTICLE IN PRESS

YPHRS-3129; No. of Pages 12

Pharmacological Research xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs

Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function Sen-Ling Feng a , Zhong-Wen Yuan a , Xiao-Jun Yao a , Wen-Zhe Ma a , Liang Liu a , Zhong-Qiu Liu b , Ying Xie a,∗ a State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute For Applied Research in Medicine and Health, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macau (SAR), China b International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, China

a r t i c l e

i n f o

Article history: Received 18 February 2016 Received in revised form 22 March 2016 Accepted 2 April 2016 Available online xxx Chemical compounds studied in this article: Tangeretin (PubChem CID: 69077) Paclitaxel (PubChem CID: 36314) Doxorubicin (PubChem CID: 31703) Docetaxel (PubChem CID: 148124) Daunorubicin (PubChem CID: 30323) 5-Fluorouracil (PubChem CID: 3385) Keywords: Tangeretin Citrus flavonoid Multidrug resistance ABCB1/P-glycoprotein

a b s t r a c t Multidrug resistance (MDR) and tumor metastasis are the main causes of chemotherapeutic treatment failure and mortality in cancer patients. In this study, at achievable nontoxic plasma concentrations, citrus flavonoid tangeretin has been shown to reverse ABCB1-mediated cancer resistance to a variety of chemotherapeutic agents effectively. Co-treatment of cells with tangeretin and paclitaxel activated apoptosis as well as arrested cell cycle at G2/M-phase. Tangeretin profoundly inhibited the ABCB1 transporter activity since it significantly increased the intracellular accumulation of doxorubicin, and flutax-2 in A2780/T cells and decreased the efflux of ABCB1 substrates in Caco2 cells without altering the expression of ABCB1. Moreover, it stimulated the ATPase activity and inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner, indicating a direct interaction with the transporter. The molecular docking results indicated a favorable binding of tangeretin with the transmemberane region site 1 of homology modeled ABCB1 transporter. The overall results demonstrated that tangeretin could sensitize ABCB1-overexpressing cancer cells to chemotherapeutical agents by directly inhibiting ABCB1 transporter function, which encouraged further animal and clinical studies in the treatment of resistant cancers. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Multidrug resistance (MDR) is the major factor leading to the failure of cancer chemotherapy, and reversing MDR has been an important goal for clinical and investigational oncologists [1,2]. The most established mechanism for MDR is the overexpression of ATP-binding cassette (ABC) family membrane transporters. Up to now, ABC transporters have 49 members [3], among which ABCB1, ABCG2 and ABCCs are known as the most important members that result in MDR in cancer cells [4,5]. ABCB1, also known as glycoprotein P (P-gp) encoded by MDR1 gene, was the first cloned human ABC transporter that can transport

Abbreviations: MDR, Multidrug resistance; P-gp, glycoprotein P; ABC, ATPbinding cassette; PTX, paclitaxel; DOX, doxorubicin; NSCLC, non-small cell lung cancer; CI, combination index; Rho 123, rhodamin123. ∗ Corresponding author. E-mail address: [email protected] (Y. Xie).

a large number of compounds including most chemotherapeutic drugs such as taxanes (e.g. paclitaxel (PTX), docetaxel) and anthracyclines (e.g. doxorubicin (DOX) and mitoxantrone) [2,6]. In cancerous tissue, the highest expression of ABCB1 was conventionally found in tumors that are derived from tissues that normally express ABCB1, such as epithelial cells of the colon, kidney, adrenal, pancreas, and liver, resulting in chemotherapeutic drug resistance [7,8]. Developing inhibitors that either down-regulate the expression of ABC proteins or inhibit the efflux function of ABC transporters would have potential clinical benefit. However, the first, second and third generation of ABC modulators such as quinine, verapamil, cyclosporine-A, tariquitor, PSC 833, LY335979, and GF120918 required high doses to reverse MDR and were associated with adverse effects [8]. These limitations have spurred efforts to search for new, multi-target compounds from natural products with higher efficacy and lower toxicity [9,10]. Flavonoids are a large group of poly-phenolic antioxidants found in fruits and vegetables. Many evidence indicated that flavonoids interact with ABC transporters and modulate MDR in tumors

http://dx.doi.org/10.1016/j.phrs.2016.04.003 1043-6618/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003

G Model YPHRS-3129; No. of Pages 12 2

ARTICLE IN PRESS S.-L. Feng et al. / Pharmacological Research xxx (2016) xxx–xxx

Fig. 1. Chemical structure of tangeretin.

[9,11–13]. In previous studies, we screened a self-built library comprised of flavonoids from natural products against human ovarian paclitaxel resistance cancer cell A2780/T to identify the most suitable candidates. Tangeretin (5,6,7,8,4 -pentamethoxyflavone; Fig. 1) was identified as one of the most effective MDR reversing agents. Tangeretin is a non-toxic dietary bioflavonoid found in citrus peel (Citrus sinensis) as well as orange juice [14,15]. It has been reported to exhibit biological effects via its anti-inflammatory, anti-tumor, cholesterol lowering, and neuroprotective activities [16–18]. As a potent chemopreventive agent, tangeretin inhibited the growth of several prostate cancer cell lines with IC50 values around 50 ␮M through induction of G0 /G1 phase cell-cycle arrest [19,20]. Moreover, in combination, tangeretin synergisti-

Fig. 2. The effect of tangeretin (TG) on the sensitivity of resistant cells to paclitaxel (PTX) (A) Tangeretin reduces the IC50 of paclitaxel in resistant cancer cells (A2780/T) but not in drug sensitive (A2780) (B). (C) Tangeretin reduces the IC50 of paclitaxel in resistant cancer cells (A549/T). Cells were treated with the indicated drugs for 48 h and subjected to SRB assay. (D) Colony formation assay of PTX in the presence or absence of tangeretin. Colony numbers were counted after Giemsa staining using the software of Quantity one-Colony counting. IC50 values are represented as means ±SD of three independent experiments performed in triplicate. ## or **, P < 0.01., ### or ***, P < 0.001, significantly different from those obtained in the absence of tangeretin.

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003

G Model

ARTICLE IN PRESS

YPHRS-3129; No. of Pages 12

S.-L. Feng et al. / Pharmacological Research xxx (2016) xxx–xxx Table 1 Tangeretin reverses the ABCB1-mediated drug resistance to chemotherapeutic agents in A2780/T and A549/T cells. Drug

A2780/T

A549/T

IC50 ± SD (␮M)

fold reversal

IC50 ± SD (␮M)

fold reversal

Paclitaxel +0.84 ␮M TG +5.21 ␮M TG +7.53 ␮M TG

2.51 1.58 0.45 0.14

± ± ± ±

0.08 0.04 0.07* 0.02**

1.00 1.59 5.66 18.88

2.53 1.03 0.27 0.03

± ± ± ±

0.41 0.33* 0.02** 0.01***

1.00 2.67 9.52 80.05

Docetaxel +0.84 ␮M TG +5.21 ␮M TG +7.53 ␮M TG

28.37 14.22 3.57 0.84

± ± ± ±

4.59 2.31* 0.58** 0.07***

1.00 1.99 7.95 33.66

12.68 7.80 3.36 1.06

± ± ± ±

2.06 1.29 0.28* 0.08**

1.00 1.58 3.82 12.07

Doxorubicin +0.84 ␮M TG +5.21 ␮M TG +7.53 ␮M TG

6.35 3.98 2.00 0.63

± ± ± ±

1.03 0.65 0.33* 0.11**

1.00 1.58 3.16 10.00

7.13 4.74 2.38 1.06

± ± ± ±

1.15 0.38 0.19* 0.08**

1.00 1.50 2.99 6.79

Daunorubicin +0.84 ␮M TG +5.21 ␮M TG +7.53 ␮M TG

7.51 5.66 3.4 0.96

± ± ± ±

0.61 0.92 0.82* 0.23**

1.00 1.34 2.25 8.03

6.70 4.74 2.99 1.06

± ± ± ±

0.54 0.38 0.24* 0.08**

1.00 1.41 2.25 6.36

5-Fluorouracil +0.83 ␮M TG +2.51 ␮M TG +7.53 ␮M TG

179.01 151.86 84.28 28.37

± ± ± ±

29.02 36.73 6.56* 4.60*

1.00 1.19 2.12 6.31

142.19 106.1 66.95 23.76

± ± ± ±

23.05 8.63 5.44* 1.93**

1.00 1.34 2.12 6.05

* ** ***

P 0.05. P 0.01. P 0.001.

cally increases the cytotoxic effect of cisplatin or doxorubicin by down-regulation of PI3 K/Akt [21] or cell cycle arrest [22]. In addition, literatures reported that tangeretin could increase the uptake of [3 H] vinblastine in Caco-2 cells at concentration of 100 ␮M [23,24] and the uptake of [3 H] vincristine in K562/ADM cells at concentration of 20 ␮M [25], indicating the potential of ABCB1 transporter inhibiting effect. However, considering its apoptosisinducing activity, how and to what extent tangeretin inhibits ABCB1 transporter in MDR cancer cell lines, and whether this activity contributes to MDR reversal are still elusive. Thus, the objectives of this investigation are to determine the effects of tangeretin on ABCB1 mediated MDR at nontoxic concentrations and to illustrate the underlying mechanism(s).

3

Scotland) at 37 ◦ C in a humidified 5% CO2 atmosphere. The indicated concentration of paclitaxel (0.94 ␮M) was added to the culture medium to maintain drug resistance for A2780/T and A549/T. The ABCB1 mRNA expression did not change significantly after grown in drug- free culture medium for 10 days for both resistant cell lines. The human colon carcinoma cell line Caco-2 was purchased from the ATCC, and cells at passage numbers 25–35 were used for the assays.

2.2. Cell cytotoxicity assay Sulphorhodamine B (SRB) assays were carried out as previously described to assess cell density, based on sensitive measure of total cellular protein [26]. Briefly, cells were seeded into flat bottomed 96-well plates at an initial density of 7.5 × 103 per well before treatment. For testing synergistic effect on the growth inhibition, cells were exposed to varying concentrations of tangeretin (7.53, 2.51 and 0.83 ␮M) and combined them with varying concentrations of PTX (1 ␮M to 0.03 nM, 10 ␮M to 0.3 nM, or 100 ␮M to 3 nM with 3.16 fold diluted respectively). After removing the medium, cells were fixed in 10% trichloroacetic acid for 1 h at 4 ◦ C, followed by tap water wash. After drying the fixed cell was stained with 0.4% SRB dissolved in 1% v/v acetic acid, washed with 1% acetic acid and dried. Then, the cells were dissolved with 10 mM Tris buffer and later measured by reading on a plate reader (Spectra MAX 250; Molecular Devices, Sunnyvale, CA) at wavelengths 490 nm. The calculation of resistance was estimated by comparing the IC50 (concentration of 50% inhibition) for the MDR cells to that of parent sensitive cells, while, the degree of reversal of MDR was calculated by dividing the IC50 for cells with the chemotherapeutic drugs in the absence of tangeretin by that obtained in the presence of tangeretin. Long-term cell survival was evaluated by colony formation assays. In brief, A2780/T or A549/T cells (1200 cells/well) in 6well plates were treated with culture medium (containing 0.94 ␮M paclitaxel for maintaining resistance) or combined with tangeretin in different concentration (containing 0.94 ␮M paclitaxel) for 8–12 days. Subsequently, the colonies were stained with 0.5% crystal violet. After rinsed with phosphate buffered saline (PBS), the colony numbers were counted using the software of Quantity one-Colony counting.

2. Materials and methods 2.3. Cell cycle analysis 2.1. Reagents and cell culture Tangeretin was purchased from Dalian Meilun Biology Technology Co., Ltd, and the structure and purity was confirmed by LC–MS in our lab. PTX, DOX, verapamil, quinidine, dimethyl sulfoxide (DMSO), RNase A, leupeptin, aprotinin, phenyl methyl sulfonyl fluoride, Triton X-100, propidium iodide (PI) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Flutax-2 was purchased from Life Technologies. Stock solutions of tangeretin (40 mM), DOX (40 mM) and PTX (80 mM) were prepared in DMSO and appropriate working concentrations were prepared in cell culture medium immediately before use. The RPMI 1640 medium, fetal bovine serum, penicillin and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY). Actin antibody was purchased from Santa Cruz Biotechnology, USA; P-gp and P53 antibodies were purchased from Calbiochem and Abcam. Human ovarian cancer cells A2780 and its PTX-resistant cell line A2780/T, human non-small cell lung cancer (NSCLC) A549 and its PTX-resistant cell line A549/T were generously provided by Professor Zhi-Hong Jiang (Macau University of science and technology, Macau). Cells were grown as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum (GIBCO, Paisley,

After 24 h, 48 h, or 72 h treatment, the cells were harvested and washed twice with ice-cold PBS. Then the cells were collected and fixed in 70% ice-cold ethanol overnight at −20 ◦ C for 2 h. Cells were re-suspended in PBS containing PI (50 ␮l/ml) and RNase A (250 ␮g/ml) for 30 min at room temperature for staining. Then they were pelleted, washed and suspended in PBS to a final concentration of 1 × 106 /ml and analyzed by flow cytometry (BD FACS Aria, BD Biosciences, San Jose, CA).

2.4. Apoptosis analysis with Annexin-V/PI double-staining assay After treatment, 1 × 106 cells were harvested, washed twice with PBS and re-suspended in 100 ␮l binding buffer (10 mM HEPES/NaOH, 140 mM NaCl and 2.5 mM CaCl2 , pH 7.4). FITC (fluorescein isothiocyanate)-Annexin V (5 ␮l) was added to the cells followed by the addition of 5 ␮l of PI (50 ␮g/ml of PBS). The samples were incubated for 15 min in the dark at 4 ◦ C and then analyzed by fluorescence-activated cell sorting cater-plus flow cytometry. Data acquisition and analysis were performed in BD FACS Aria with FlowJo software.

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003

G Model YPHRS-3129; No. of Pages 12 4

ARTICLE IN PRESS S.-L. Feng et al. / Pharmacological Research xxx (2016) xxx–xxx

Fig. 3. Effect of the Tangeretin-PTX combination on apoptotic induction in A2780 and A2780/T cells. The cells were treated with PTX and/or tangeretin (TG) for 24 h, 48 h, 72 h and strained with PI and fluorescein isothiocyanate-conjugated annexin V. The graphs show the percentage of proportion of apoptosis cells. The data are representative of three different experiments and are shown as mean ±SD (n = 3). ## or **, P < 0.01., ### or ***, P < 0.001, significantly different from those obtained in the absence of tangeretin.

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003

G Model YPHRS-3129; No. of Pages 12

ARTICLE IN PRESS S.-L. Feng et al. / Pharmacological Research xxx (2016) xxx–xxx

5

Fig. 4. Effect of the combination tangeretin-PTX on cell cycle in A2780 and A2780/T cells. The cells were treated with PTX and tangeretin (TG) for 48 h and then fixed and stained with PI. The cell cycle distribution profiles of the cells were determined by flow cytometry. And the graph shows the distribution of cells in the cell cycle. Representative histograms indicate the percentages of cells in the G0/G1,S and G2/M phases of the cell cycle with the average values (±S.D.). Data are representative of three different experiments. Tangeretin-PTX combination induces G2/M arrest in A2780/T cells. ## or **, P < 0.01., ### or ***, P < 0.001.

2.5. Drug combination assay The Chou-Talalay Method were used to study the synergistic therapeutic effect of tangeretin in combination of PTX [27]. Drug resistant A2780/T cells were treated with compounds alone or a serially diluted mixture of tangeretin (IC50 = 36.35 ␮M) and PTX (IC50 = 2.51 ␮M) for 48 h. The 2-fold serial solutions with several concentration points above and below its IC50 value was used for evaluating cytotoxicity of combination by SRB assay. Data were expressed as Combination index (CI), which offers definition for addictive (CI = 1), synergism (CI < 1) and antagonism (CI > 1) in drug combination. With the use of CalcuSyn software

v. 2.1 (Bio-soft, Cambridge, UK), synergy is further refined as synergism (combination index = 0.3–0.7), strong synergism (combination index = 0.1–0.3), and very strong synergism (combination index < 0.1) [28]. 2.6. Intracellular uptake and accumulation assay Drug uptake assay were determined by fluorescence microscopy. A2780 or A2780/T cells (5 × 106 ) were seeded on the cover glass (ISO LAB 20 × 20 mm). DOX (5 ␮M), or flutax-2 (1 ␮M) (an active fluorescent taxoid) alone or in combination with tangeretin (7.53 ␮M) was added and incubated for 8 h. After

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003

G Model YPHRS-3129; No. of Pages 12 6

ARTICLE IN PRESS S.-L. Feng et al. / Pharmacological Research xxx (2016) xxx–xxx

Fig. 5. Effect of tangeretin (TG) on intracellular accumulation of doxorubicin (DOX) and flutax-2(F-tax) in drug-resistant ovarian cancer cells. A2780 cells or A2780/T Cells treated with 5 ␮M DOX (A and B) or 5 ␮M F-tax (C and D) for 8 h in the absence or presence of 7.53 ␮M tangeretin, and 20 ␮M quinidine (positive control) as indicated. Intracellular DOX and F-tax accumulation were observed with a florescence microscope (A and C) or evaluated by measuring florescence with flow cytometry (B and D) as described in Section 2 The experiments were repeated for at least 3 times, presented are representative images.

treatment, cells were washed and fixed in 4 wt% formaldehyde (Sigma-Aldrich). 1 ␮g/ml blue-fluorescent DAPI (1 mg/ml in H2 O stock solution; Invitrogen D1306) was used to stain nuclear DNA. One drop of fluorescent preservation solution (fluorsave reagent, CALBIOCHEM) was added before observation. Imaging was acquired for comparing the intracellular amount of DOX and flutax-2 by a Fluorescence Microscopy (Leica DM2500, Leica, Geman). For accumulation assay with flow cytometry, A2780 or A2780/T cells were incubated with flutax-2 (1 ␮M) or DOX (5 ␮M) in the presence or absence of tangeretin (7.53 ␮M) for 8 h. After incu-

bation, cells were collected, re-suspended in 500 ␮l of PBS after washed twice, and analyzed by flow cytometry (BD FACS Aria, BD Biosciences, San Jose, CA). The intracellular fluorescence was measured with excitation and emission wavelengths (nm) of 480–585 and 496–524 for DOX and flutax-2, respectively. Quinidine (QND, 20 ␮M) was used as a positive control of ABCB1 inhibitor. 2.7. Transport studies in Caco-2 monolayer model For the bi-directional transport studies [29], Caco-2 cells were seeded on Transwell® insert (Corning Inc., MA) 12-well

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003

G Model YPHRS-3129; No. of Pages 12

ARTICLE IN PRESS S.-L. Feng et al. / Pharmacological Research xxx (2016) xxx–xxx

7

Fig. 6. Tangeretin (TG) increased the absorption and decreased the efflux ratio of DOX in Caco-2 cells. (A) The effects of tangeretin on the directional transport of DOX (10 ␮M) across Caco-2 cell monolayers. (B) The effects of tangeretin on the efflux ratio of DOX (10 ␮M) in Caco-2 cell monolayers. Data represent the mean ±SD of three individual determinations. 䊐 AP → BL transport, 䊏 BL → AP transport. ## or **, P < 0.01., ### or ***, P < 0.001, significantly different from those obtained in the absence of tangeretin.

plates and formed a confluent monolayer over 21 days. The quality of the monolayers was assessed by measuring their transepithelial electrical resistance (TEER) before and after the transport experiments using a WPI EVOM volt-ohmmeter fitted with STX2 chopstick electrodes (World Precision Instruments, Sarasota, FL). Lucifer yellow across the cell layer was determined at the end of each experiment and the permeability was<1% in all of the conducted experiments. Briefly, after pre-incubation with HBSS buffer, rhodamin123 (Rho123, 5 ␮M) or DOX (10 ␮M) loading solution was added to the donor side (Apical/Basolateral of the insert). The samples (100 ␮l) were collected from receptor side (Basolateral or Apical) at predetermined time intervals of 30, 60, 90, and 120 min, and immediately detected for the fluorescence intensity in 96 well black plate (Corning; Cat. 3603) by a microplate reader (infinite M200 PRO, TECAN, Switzerland). An equal volume of blank HBSS was replaced to the receptor side immediately after each sampling. For inhibition studies, bidirectional transport of target compound was conducted with tangeretin added in both apical (AP) and basolateral (BL) chambers. Quinidine (QND) was used as a positive control ABCB1 inhibitor. The apparent permeability coefficients (Papp) were calculated as Papp =

1 dQ × C0 A dt

where dQ/dt (mM/s) is the rate of permeation of compound across the cells, C0 (mM) is the donor compartment concentration at time zero and A (cm2 ) is the area of the cell monolayer. The change in Efflux Ratio (ER = Papp (BL to AP)/Papp (AP to BL)) in the presence of tangeretin and putative inhibitor QND was used for assessing their relative inhibitory potency to ABCB1 transporter.

2.8. ABCB1 ATPase activity assay The effect of tangeretin on ABCB1 ATPase activity as well as the inhibitory effect against verapamil-stimulated ABCB1 ATPase activity was estimated by Pgp-GloTM assay systems Promega, USA Sodium orthovanadate (Na3 VO4 ) and verapamil were used as control to demonstrate drug-inhibited and drug-stimulated activity of Pgp ATPase, respectively. Following manufacture’s instruction, 0.25 mM Na3 VO4 , 0.5 mM verapamil, or tangeretin in various concentrations was incubated with assay buffer, 25 ␮g recombinant human ABCB1 membranes and 5 mM MgATP at 37 ◦ C for 40 min. For examination the inhibitory effects of tangeretin against verapamilstimulated Pgp ATPase activity, then 200 ␮M verapamil was added with tangeretin together. Then, ATP detection buffer was added to stop reaction and initiate luminescence. After reaction for 20 min, the white opaque 96-well plate (corning, USA) was read on luminometer (infinite M200 PRO,TECAN, Switzerland). The changes of relative light units (RLU) were determined by comparing Na3 VO4 -treated samples with tangeretin only or tangeretin and verapamil combination-treated samples, and thereafter, the ATP consumed was measured by comparing to a standard curve.

2.9. Reverse transcription (RT)-PCR analysis RT-PCR was performed to evaluate MDR1 mRNA expression. mRNA from cell lysates were purified by using oligo (dT) magnetic beads (Life technologies) and reverse transcribed by using SuperScript II (Life technologies) following the manufacturer’s protocol. The primer sequences: 5 -GAGAGATCCTCACCAAGCGG3 and 3 -CGAGCCTGGTAGTCAATGCT-5 for MDR1, and 5 -AGAAGGCTGGGGCTCATTTG-3 and 3 -AGGGGCCATC-

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003

G Model YPHRS-3129; No. of Pages 12

ARTICLE IN PRESS S.-L. Feng et al. / Pharmacological Research xxx (2016) xxx–xxx

8

Fig. 7. The capacity of tangeretin (TG) on stimulating ABCB1 ATPase activity and on inhibiting 200 ␮M verapamil-stimulated ABCB1 ATPase activity. (A) EC50 measurements for stimulating ABCB1 ATPase activity by tangeretin; (B) IC50 measurements for inhibiting 200 ␮M verapamil-stimulated ABCB1 ATPase activity by tangeretin. Luminescence was read on a luminometer and data was analyzed as described in Section 2.

Fig. 8. Effect of tangeretin (TG) on ABCB1 and p53 expression in MDR ovarian cancer cells. A2780/T cells or A2780 cells were treated with tangeretin at various concentrations for 48 h. (A) The MDR1 mRNA level was determined by RT-PCR (B) Equal amounts of total lysate were loaded and detected by Western blot. Tangeretin did not influence either ABCB1 mRNA or protein expression levels, but up regulated the p53 expression. The experiments were performed three times.

2.11. Molecular modeling—ABCB1 CACAGTCTTC-5 for control gene eukaryotic translation initiation factor (TIF) [26]. Quantitative RT-PCR analysis was performed using SYBR Green (Molecular Probes) reagents and ViiATM 7 Real-Time PCR System (Life technologies). Gene expression levels were calculated from cycle threshold (Ct) values and normalized with control gene TIF.

2.10. Western blot analysis Treated cells were harvested and rinsed twice with ice-cold PBS buffer. Then cells were lysed in RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride and EDTA) containing protease inhibitor cocktails (Roche Life Science, USA). Protein concentration was determined using the BCA protein assay kit. Equal amounts of total cell lysates were resolved using SDS-PAGE and transferred onto PVDF membranes (Millipore, Darmstadt, Germany). After incubation in a blocking solution containing with 5% (w/v) skim milk (Nestle Carnation, New Zealand) in TBST buffer (10 mM tris-buffered saline and 0.1% Tween20) for 2 h at room temperature, the membranes were immunoblotted overnight with primary and secondary antibodies and subsequently visualized with an enhanced chemiluminescence detection kit (Thermo ScientificTM SuperSignalTM West Pico Chemiluminescent Substrate, USA). ␤-actin was used as the loading control for the experimental data analysis.

In order to figure out the exact binding site for tangeretin, we used homology modeling and molecular docking to study the interaction between human P-Glycoprotein and tangeretin. Human P-glycoprotein (ABCB1) was thought to have four sites interacting with the inhibitors [30,31], so we rebuilt the four sites using Prime v2.1 in Maestro 9.0 (Schrodinger, Inc., New York, NY, 2009). The 3D structures of ABCB1 from the mouse was selected as the templates: The complex structure cocrystallized with QZ59RRR (PDB: 4M2S) for site 1, the complex structure cocrystallized with QZ59-SSS (PDB: 4M2T) for site 2, the apo structure (PDB: 3G5U) for site3 and site 4. The ligands from the complex templates were retained and used to define the site 1 and site 2 in the homology structures. The site 3 was defined by residues contributing to verapamil binding and the site 4 was defined by two residues which were common to the other three sites [32]. All the docking calculations for four sites were performed in the Induced Fit Docking module (Schrodinger, Inc., New York, NY, 2009) and the pose was ranked by the XP mode of Glide program v5.5 (Schrodinger, Inc., New York, NY, 2009). Then we selected the pose with the highest docking for further conformational analysis. 2.12. Statistical analysis All experiments were repeated at least three times and data are expressed as the mean ± SD. Microsoft Excel 2010 and GraphPad Prism 5.00 software were used in data processing and analyzing. Statistical analysis was carried out using Student’s t-test or one-way analysis of variance. And the statistical significance was determined to be P < 0.05 (*), P < 0.01 (**) or P < 0.001(***).

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003

G Model

ARTICLE IN PRESS

YPHRS-3129; No. of Pages 12

S.-L. Feng et al. / Pharmacological Research xxx (2016) xxx–xxx

9

Fig. 9. Docking analysis of tangeretin with human ABCB1 homology model. (A) The cartoon style of the homology model of human ABCB1.The binding poses of QZ59RRR(green) and tangeretin (orange) are shown in site 1. (B) The interaction between tangeretin and the surrounding residues. The red dotted line represents hydrogen bond between atoms, while the yellow dotted line represents ␲-␲ stacking between aromatic rings. Important amino acids are depicted as sticks with the atoms colored as carbon—green, hydrogen—white, nitrogen—blue, oxygen—red, whereas tangeretin is shown with the same color scheme as above except carbon atoms are represented in orange. (C) A interaction sketch between tangeretin and its binding site. Residues are shown as colored bubbles, cyan indicates polar and green indicates hydrophobic residues. Hydrogen bond is shown by purple dotted arrow.

Table 2 The values of CI and the synergism dose of TG and PTX at Fa 0.5 (ED50). Data for Fa = 0.5

CI value

Dose TG (␮M)

Dose PTX (␮M)

Tangeretin PTX Tangeretin and PTX

– – 0.098

67.45 – 2.84

– 3.61 0.20

Note: CI analyses of the effects of tangeretin in combination with paclitaxel are shown. The CI values were plotted as a function of the particular inhibitory effect. CI values <1 represent a synergistic combination, CI values equal to 1 are additive and CI values >1 represent antagonistic combinations. We conclude from Table that PTX was significantly reduced in tangeretin treated A2780/T cells.

3. Results 3.1. Tangeretin enhances the efficacy of chemotherapeutic agents in ABCB1 overexpressing cells The stably paclitaxel-resistant cell line (A2780/T, cultured with 0.94 ␮M PTX to maintain drug resistance) exerted much higher tolerance than their parental sensitive cell line (A2780). The mean IC50 values for PTX and DOX were 501-fold and 158- fold greater in A2780/T cells than that of A2780, respectively. The intrinsic cytotoxicity of tangeretin was also evaluated and similar IC50 was observed for A2780 and A2780/T (culture medium without 0.94 ␮M PTX) with mean values of 35.57 and 36.35 ␮M, respectively. Moreover, the results showed that tangeretin at 7.53 ␮M, had no obvious cytotoxic effect to all cell lines, and more than 90% cells survived. Therefore, tangeretin was tested in the reversal assays at a maximum concentration of 7.53 ␮M.

Next, we evaluated whether tangeretin could reversal ABCB1 mediated drug resistance in A2780 and A2780/T. Tangeretin significantly decreased the IC50 of PTX in A2780/T cell lines in a concentration-dependent manner, as shown by a shift in the cytotoxicity curves to the left in Fig. 2A. As shown in Table 1, treatment with 0.83, 2.51, and 7.53 ␮M of tangeretin significantly increased the sensitivity of A2780/T cells to PTX with reversal fold of 1.59, 5.66-, and 18.88-, respectively. However, no sensitized effect of tangeretin was observed in the parental A2780 cells (Fig. 2B). Moreover, at concentration of 7.53 ␮M, tangeretin also reduced the IC50 values of docetaxel, DOX, and daunorubicin with reversal fold of 33.66-, 10.00- and 8.03-, respectively, whereas it also slightly decreased the IC50 values of 5-fluorouracil (non-substrate of ABCB1) with reversal fold of 6.31 as shown in Table 1. Furthermore, we observed that similar reversal effects of tangeretin to chemotherapeutics in another ABCB1-overexpressing non-small cell human lung cancer cell line A549/T (PTX-resistance) as shown in Fig. 2C. Tangeretin (0.84, 2.51, and 7.53 ␮M) was found to enhance the cytotoxicity of PTX (2.67-, 9.52-, and 80.05-fold), docetaxel (1.58-, 3.82-, and 12.07-fold), DOX (1.50-, 2.99-, 6.79fold), and daunorubicin (1.41-, 2.25-, 6.36-fold) in a concentration dependent manner (Table 1). These results suggest that tangeretin significantly sensitizes ABCB1-overexpressing cells to chemotherapeutic drugs. We also investigated the long time reversal effects of tangeretin on ABCB1 mediated MDR to PTX by using colony formation assay. Significant inhibition of cell colony formation was observed in the presence of tangeretin in combination with PTX, whereas inhibition effect was not observed with either tangeretin or PTX alone (Fig. 2D). Therefore, all these results indicate that combination of

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003

G Model YPHRS-3129; No. of Pages 12 10

ARTICLE IN PRESS S.-L. Feng et al. / Pharmacological Research xxx (2016) xxx–xxx

tangeretin with PTX elicits significantly higher cytotoxic response in ABCB1 overexpression cancer cells.

3.2. Tangeretin potentiates the apoptosis induced by PTX As MDR was mediated by increased to resistance to anticancer drug-induced apoptosis, we next investigated whether tangeretin increased the PTX-induced apoptosis in A2780 and A2780/T cells by using flow cytometry. Consistent with the ability to inhibit cell growth, significantly higher rates of apoptosis were induced in A2780/T cells by tangeretin combined with PTX as shown in Fig. 3. We found that treatment with only 0.83 ␮M of tangeretin could boost apoptosis induced by PTX (0.94 ␮M) to a similar level as that of 2.51 ␮M of PTX (IC50 ). However single treatment of tangeretin or PTX did not show any apoptosis induction. To further confirm these results, we examined the expression level of p53, the well-established biochemical markers of cell cycle and apoptosis. Consistent with cell growth inhibition and apoptosis, treatment of PTX in combination with tangeretin resulted in accumulation of p53 protein in treated cells (Fig. 8).

3.5. Tangeretin increases the intracellular accumulation of DOX and Flutax-2 The results above proved that tangeretin had a significant effect on reversing MDR in A2780/T and A549/T cells to chemotherapeutic agents such as PTX. However, the mechanism of this phenomenon is unknown. Therefore, we conducted assays to examine the effect of tangeretin on the accumulation of DOX, and Flutax-2 (a fluorescent taxol derivative) in A2780 cells and their corresponding MDR A2780/T cells by using fluorescence microscope and flow cytometry analysis. In absence of tangeretin, the fluorescence of DOX and Flutax-2 was significantly higher in the parental sensitive cells than the MDR cells (Fig. 5). However, tangeretin (7.53 ␮M) and QND (positive control, 20 ␮M) significantly increased the intracellular accumulation of DOX (Fig. 5A), and Flutax-2 (Fig. 5C) without affecting their parental A2780 cells. Enhanced intracellular accumulation of DOX, or Flutax-2 by tangeretin were further confirmed with flow cytometry analysis, as shown in Fig. 5B and D. Taken together, these results showed that tangeretin significantly increased the intracellular accumulation of chemotherapeutic drugs in MDR cells, thus increased their cytotoxicity to these MDR cells.

3.3. Tangeretin-PTX combination arrests resistant cells at the G2/M-phase

3.6. Tangeretin inhibits the efflux activity of ABCB1 transporter in Caco-2 cells

In this experiment, asynchronously growing A2780/T cells and its sensitive parental cell line A2780, treated with PTX in the absence or presence of tangeretin, were examined for their cell cycle progressions by flow cytometry. In untreated control, the percentage of A2780 cells in G0/G1-, S- and G2/M-phases were 62.23%, 19.23% and 16.90%, respectively, the percentage of A2780/T cells in G0/G1-, S- and G2/M-phases were 66.40%, 14.10% and 16.73%, respectively. Single exposure (24, 48, and 72 h) with PTX (0.01 ␮M) to A2780 cells resulted in G2-M arrest (>60%), manifested by an increased G2-M content, and decreased G1 phase content (Fig. 4). After incubation with 0.94 ␮M of PTX alone, there were 68.30% G1 phase and 18.87% G2 phase A2780/T cells which was same as none treatment. However, this distribution significantly shifted to 13.87% G1 and 72.37% G2 phase after co-treatment of 7.53 ␮M tangeretin and 0.94 ␮M PTX in A2780/T cells for 48 h (Fig. 4). This pattern was evident at 24 h and persisted over 72 h after treatment. Even at the lowest concentration of tangeretin (0.83 ␮M), a pronounced G2/M arrest was observed. But tangeretin (7.53 ␮M) alone had no effect on cell cycle distribution of A2780/T. Thus, while A2780/T cells were remarkably resistant to PTX, combination of tangeretin and PTX could greatly increase the accumulation of G2/M-phase cells (>70%) which was largely at the expense of cells failing to cycle into G1-phase.

Caco-2 cells derived from a human colorectal carcinoma are widely used as an in vitro model for predicting human drug absorption and efflux activity of transporters [29]. To further confirm the effect of tangeretin on ABCB1 transporter function, we evaluated the Papp and Efflux Ratio (the ratio between the Papp from the BL to the AP side and that from the AP to the BL side) of the ABCB1 substrates Rho 123, and DOX in the presence or absence of tangeretin in Caco-2 monolayer cell model. Two hours after administration, the Papp (A–B) values of Rho 123 (Fig. 6A), and DOX (Fig. 6A) was increased from A to B side in the presence of tangeretin dosedependently; moreover, the efflux ratios of Rho 123 (Fig. 6A), and DOX (Fig. 6A) was significantly decreased to around 1.0 in the presence of 7.53 ␮M tangeretin with inhibition >70%. Intriguingly, the inhibitory effect of tangeretin at 2.51 ␮M was stronger than that of 20 ␮M of QND (positive control). These results were in agreement with the notion that tangeretin increased Rho 123 and DOX accumulation by inhibiting ABCB1 transporter.

3.4. Tangeretin exerts synergistic effect combining with PTX in MDR cells The combination cytotoxic effect of tangeretin with PTX in A2780/T cells was evaluated using the Median Effect method. The combination index (CI) values computed at 50% and 90% cell kill were 0.098(CI at ED50 ) and 0.003 (CI at ED90 ) indicating strong synergistic cytotoxic effect (CI < 0.1) for combinations of tangeretin with PTX in MDR A2780/T cells. With CalcuSyn simulation, an ED50 is produced by 67.45 ␮M tangeretin or 3.61 ␮M PTX alone. However, in combination with 2.84 ␮M tangeretin, ED50 of PTX is decreased to 0.20 ␮M which is an 18-fold decrease for the ED50 value (Table 2).

3.7. Tangeretin activates the ABCB1 ATPase activity ATP hydrolysis is the energy source for coupled substrate transport by the ABC transporters against a concentration gradient, which is proportional to the transporter activity. Therefore, we measured ABCB1-mediated ATP hydrolysis with different concentrations of tangeretin. As shown in Fig. 7A, tangeretin produced up to a maximal stimulation of 3-fold of the ABCB1 ATPase activity in a dose-dependent manner with EC50 at 11.57 ␮M, suggesting that tangeretin affects the ATPase activity of ABCB1 and may interact at the drug-substrate-binding site as a substrate of ABCB1. The effects of tangeretin on verapamil stimulated ABCB1 ATPase activities were examined to characterize inhibition effect of tangeretin on ABCB1 ATPase activity. As a substrate for transport, verapamil is sometimes referred as an ABCB1 inhibitor because it inhibits ABCB1 activity with other substrates by interfering with their transport in a competitive mode. We found that tangeretin significant reduced the 200 ␮M verapamil-stimulated ATPase activity with an IC50 value of 7.87 ␮M in Fig. 7B, indicating it is an ABCB1 ATPase inhibitor.

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003

G Model YPHRS-3129; No. of Pages 12

ARTICLE IN PRESS S.-L. Feng et al. / Pharmacological Research xxx (2016) xxx–xxx

3.8. Tangeretin does not affect the expression of ABCB1 To further understanding the mechanism, we also determined the effect of tangeretin on the expression of ABCB1 at both mRNA and protein levels since down-regulation of drug transporter could increase the sensitivity of cancer cells to chemotherapeutics. At the reversal concentrations (0.83–7.53 ␮M), tangeretin did not alter the mRNA (Fig. 8A) or protein level of ABCB1 (Fig. 8B) in A2780/T cells. These findings revealed that the MDR reversal effect of tangeretin was not due to inhibition of ABCB1 expression. Therefore, the mechanism of the sensitization effect of tangeretin in MDR cells seems to be via inhibiting ABCB1 transporter function leading to an increase in intracellular accumulation of chemotherapeutic drugs. 3.9. Molecular docking simulation of tangeretin within drug binding cavity of ABCB1 In order to understand the binding mechanism of tangeretin to homology model of human ABCB1 at molecular level, ABCB1-QZ59RRR (site-1), ABCB1-QZ59-SSS (site-2), ABCB1-verapamil (site-3), and site common to above three sites (site-4) as well as ATP binding site were used for glide docking. According to the docking result, the pose of tangeretin was only accommodated to site 1 with Docking score (kcal/mol) at −9.216. There was no pose suitable for tangeretin to bind other three sites. Thus, site 1 was the only rational site for tangeretin. As shown in Fig. 9, the binding site of tangeretin was partially superposed with the binding site of QZ59-RRR known as site 1.The ring-a of tangeretin substituted with four methoxyl groups was mainly engaged in hydrophobic contacts with Tyr307, Phe303, Tyr310, Phe335, Leu339, Leu336,Leu332. The carbonyl of ring-b formed two hydrogen bonds with Tyr 307 and Gln725, which also appeared in the binding of vardenafil and tadalafil [30]. As for ring-c, the ␲-␲ stacking with Phe728 and the hydrophobic contacts with Tyr953, Met75, Val71, which kept the conformation stable. 4. Discussion Chemotherapeutic agents such as PTX, DOX are widely used for treatments of advanced human cancers, but long-term treatment leads to drug resistance even they are initially effective. Great efforts have been made to search for new effective resistance modulators targeting on efflux pumps with low toxicity and fewer side effects. A wide variety of phytochemicals from natural resources, such as flavonoids, have been suggested to protect us from cancer (chemopreventive) or enhance the tumoricidal effects of chemotherapy (chemosensitizers) [33,34]. Strategies using combination chemotherapeutic agents with highly promising dietary flavonoids to reverse MDR in cancer therapy represent the most useful alternatives for achieving higher curability with least toxicity. The synergistic effects of tangeretin on increasing the cytotoxic effect of cisplatin [21] or doxorubicin [22,35] have been reported at superphysiological doses (100–150 ␮M). Previous pharmacokinetic studies shown that Cmax of tangeretin was only around 1–5 ␮M in human [36] and in rat [37]. Thus the in vivo plasma concentration of tangeretin is far below the concentration (100–150 ␮M) used in these combination treatment to resistant cancer cells, indicating that these in vitro observed effects of tangeretin may not be achieved in vivo. In this study, we demonstrated that tangeretin, at achievable nontoxic plasma concentrations, significantly sensitized ABCB1ovexpressing cells to chemotherapeutic agents including DOX, PTX, docetaxel and daunorubicin for the first time. It significantly increased the cellular toxicity of ABCB1 substrates in A2780/T and A549/T cells using SRB assays and colon formation assays in a p53-dependent manner [38], but it had no effect on the IC50 of

11

the parental A2780 and A549 cells. Consistent with the results of toxicity assays, treatment with tangeretin promoted PTX-induced apoptosis and G2/M phase (>70%) cell cycle arrest. Moreover, CI values in combination study indicated a strong synergistic cytotoxic effect of combining tangeretin with PTX in ABCB1-overexpressing A2780/T cells. All these results proved that tangeretin can increase the sensitivity of ABCB1-mediated MDR cells to chemotherapeutic agents. To elaborate the mechanism of the reversal effect of tangeretin, we investigated whether tangeretin could inhibit the efflux function of ABCB1 to enhance the cytotoxicity of agents by increasing the intracellular drug concentration. It was found that tangeretin remarkably enhanced the intracellular accumulation of DOX and flutax-2 in drug resistant cells and reduced the efflux ratio of Rho123, and DOX in a Caco-2 monolayer cell model, indicating that it could inhibit the efflux activity of ABCB1 transporter. However, tangeretin did not alter the expression of MDR1 mRNA and ABCB1 protein. Thus, we presumed that the reversal effect of tangeretin may result from inhibition of the efflux function of ABCB1 transporter. As energy used by ABCB1 transporter comes from ATP hydrolysis, one of the major reasons to inhibit the activity of ABCB1 transporter activity is to deplete ATP. We investigated the effect of tangeretin on the ATPase activity of ABCB1 transporter to confirm our previous assumption. In this study, tangeretin was found to stimulate the ATPase activity of ABCB1 transporter and inhibit verapamil-stimulated ATPase activity in the same time, suggesting that it may be not only a substrate of ABCB1 transporter but also an inhibitor of ABCB1 ATPase. The substrates could be sometimes inhibitors for ABCb1 transporter such as verapamil. Although the activity of ATPase was increased, the efflux function of ABCB1 was inhibited accordingly by tangeretin. Thus we reasoned that tangeretin may competitively bound to the substrate-binding site of ABCB1, leaving little place for other substrates to bind to the transporter, which resulted in decreased activity of ABCB1 transporter as well as enhanced intracellular concentration of other substrates. To better understanding the important interactions of tangeretin interaction with the active site of ABCB1 protein, docking simulation was performed with the ATP binding site of ABCB1. The predicted binding conformation of tangeretin within the large hydrophobic drug binding cavity (site-1) of human ABCB1 is similar as the nobiletin which is also a citrus flavonoid with a structure similar to that of tangeretin. Known as polymethoxylated flavones, both nobiletin and tangeretin could modulate the function of ABCB1 transporter by binding to site-1 [39], indicating that the methoxyl and aromatic ring are important for interaction with the drugbinding cavity of ABCB1 transporters. In the case of commonly used chemotherapy drugs, numerous candidate mechanisms for acquired drug resistance had been described such as reduced apoptosis, advanced DNA damage repair mechanisms, altered drug metabolism as well as ABC efflux transporters [40]. Therefore, there may be other mechanisms that also contribute to the sensitizing effect of tangeretin in ABCB1overexpressing MDR cancer cells such as the PI3K/AKT/mTOR and MAP kinase signaling pathways [41–43]. This part of work was carrying out in our lab which may help to explain the sensitizing effect of tangeretin to 5-fluorouracil. Moreover, we are also working on the in vivo study to further demonstrate the effect of tangeretin on the MDR tumor as well as its modulation of the function of ABCB1 transporter. 5. Conclusion For the first time, this study gave evidence that tangeretin significantly enhances the efficacy of conventional chemotherapeutic agents in ABCB1-overexpressing MDR cancer cells via directly inhibiting ABCB1 transport function. Moreover, the reversal effect

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003

G Model YPHRS-3129; No. of Pages 12

ARTICLE IN PRESS S.-L. Feng et al. / Pharmacological Research xxx (2016) xxx–xxx

12

of tangeretin is independent of inhibiting ABCB1 expression. The strong synergistic effects between tangeretin and chemotherapeutic agents were demonstrated in this study at clinically achievable non-toxic concentrations, indicating that combination use of tangeretin may be a useful strategy to overcome MDR. Considering the broad-spectrum organ safety of tangeretin which has been demonstrated in animals in vivo [44], our present work should expedite the exploration and use of tangeretin in enhancing the efficacy of ABCB1 substrate chemotherapeutic agents in experimental animal studies as well as clinical trials. Conflict of interest We declare that we have no conflict of interest. Acknowledgment This work was financially supported by the Macao Science and Technology Development Fund, Macau Special Administrative Region (090/2012/A3 to Y.Xie.). References [1] H. Xing, D. Weng, G. Chen, W. Tao, T. Zhu, X. Yang, L. Meng, S. Wang, Y. Lu, D. Ma, Activation of fibronectin/PI-3K/Akt2 leads to chemoresistance to docetaxel by regulating survivin protein expression in ovarian and breast cancer cells, Cancer Lett. 261 (1) (2008) 108–119. [2] R. Geney, M. Ungureanu l, D. Li, I. Ojima, Overcoming multidrug resistance in taxane chemotherapy, Clin. Chem. Lab. Med. 40 (9) (2002) 918–925. [3] V. Vasiliou, K. Vasiliou, D.W. Nebert, Human ATP-binding cassette (ABC) transporter family, Hum. Genomics 3 (3) (2009) 281–290. [4] R. Perez-Tomas, Multidrug resistance: retrospect and prospects in anti-cancer drug treatment, Curr. Med. Chem. 13 (16) (2006) 1859–1876. [5] M.M. Gottesman, T. Fojo, S.E. Bates, Multidrug resistance in cancer: role of ATP-dependent transporters, Nat. Rev. Cancer 2 (1) (2002) 48–58. [6] L. Gianni, Anthracycline resistance: the problem and its current definition, Semin. Oncol. 24 (4 Suppl. 10) (1997), S10-11-S10-17. [7] S.V. Ambudkar, C. Kimchi-Sarfaty, Z.E. Sauna, M.M. Gottesman, P-glycoprotein: from genomics to mechanism, Oncogene 22 (47) (2003) 7468–7485. [8] H. Thomas, H.M. Coley, Overcoming multidrug resistance in cancer: an update on the clinical strategy of inhibiting p-glycoprotein, Cancer Control 10 (2) (2003) 159–165. [9] T. Bansal, M. Jaggi, R.K. Khar, S. Talegaonkar, Emerging significance of flavonoids as P-glycoprotein inhibitors in cancer chemotherapy, J. Pharm. Pharm. Sci. 12 (1) (2009) 46–78. [10] S. Karthikeyan, S.L. Hoti, Development of fourth generation ABC inhibitors from natural products: a novel approach to overcome cancer multidrug resistance, Anticancer Agents Med. Chem. 15 (5) (2015) 605–615. [11] A.I. Alvarez, R. Real, M. Perez, G. Mendoza, J.G. Prieto, G. Merino, Modulation of the activity of ABC transporters (P-glycoprotein, MRP2, BCRP) by flavonoids and drug response, J. Pharm. Sci. 99 (2) (2010) 598–617. [12] Y. Li, J.L. Revalde, G. Reid, J.W. Paxton, Interactions of dietary phytochemicals with ABC transporters: possible implications for drug disposition and multidrug resistance in cancer, Drug Metab. Rev. 42 (4) (2010) 590–611. [13] M.E. Morris, S. Zhang, Flavonoid-drug interactions: effects of flavonoids on ABC transporters, Life Sci. 78 (18) (2006) 2116–2130. [14] Y. Nogata, K. Sakamoto, H. Shiratsuchi, T. Ishii, M. Yano, H. Ohta, Flavonoid composition of fruit tissues of citrus species, Biosci. Biotechnol. Biochem. 70 (1) (2006) 178–192. [15] J. Chen, A. Creed, A.Y. Chen, H. Huang, Z. Li, G.O. Rankin, X. Ye, G. Xu, Y.C. Chen, Nobiletin suppresses cell viability through AKT pathways in PC-3 and DU-145 prostate cancer cells, BMC Pharmacol. Toxicol. 15 (2014) 59. [16] M.S. Kim, H.J. Hur, D.Y. Kwon, J.T. Hwang, Tangeretin stimulates glucose uptake via regulation of AMPK signaling pathways in C2C12 myotubes and improves glucose tolerance in high-fat diet-induced obese mice, Mol. Cell. Endocrinol. 358 (1) (2012) 127–134. [17] E. Meiyanto, A. Hermawan, Anindyajati, Natural products for cancer-targeted therapy: citrus flavonoids as potent chemopreventive agents, Asian Pac. J. Cancer Prev. 13 (2) (2012) 427–436. [18] S.C. Ho, C.C. Lin, Investigation of heat treating conditions for enhancing the anti-inflammatory activity of citrus fruit (Citrus reticulata) peels, J. Agric. Food Chem. 56 (17) (2008) 7976–7982. [19] M.H. Pan, W.J. Chen, S.Y. Lin-Shiau, C.T. Ho, J.K. Lin, Tangeretin induces cell-cycle G1 arrest through inhibiting cyclin-dependent kinases 2 and 4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal carcinoma cells, Carcinogenesis 23 (10) (2002) 1677–1684.

[20] K.L. Morley, P.J. Ferguson, J. Koropatnick, Tangeretin and nobiletin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells, Cancer Lett. 251 (1) (2007) 168–178. [21] E.S.A. Arafa, Q.Z. Zhu, B.M. Barakat, G. Wani, Q. Zhao, M.A. El-Mahdy, A.A. Wani, Tangeretin sensitizes cisplatin-resistant human ovarian cancer cells through downregulation of phosphoinositide 3-Kinase/Akt signaling pathway, Cancer Res. 69 (23) (2009) 8910–8917. [22] E. Meiyanto, A. Fitriasari, A. Hermawan, S. Junedi, R.A. Susidarti, The improvement of doxorubicin activity on breast cancer cell lines by tangeretin through cell cycle modulation, Oriental Pharm. Exp. Med. 11 (3) (2011) 183–190. [23] H. Takanaga, A. Ohnishi, S. Yamada, H. Matsuo, S. Morimoto, Y. Shoyama, H. Ohtani, Y. Sawada, Polymethoxylated flavones in orange juice are inhibitors of P-glycoprotein but not cytochrome P450 3A4, J. Pharmacol. Exp. Ther. 293 (1) (2000) 230–236. [24] Y. Honda, F. Ushigome, N. Koyabu, S. Morimoto, Y. Shoyama, T. Uchiumi, M. Kuwano, H. Ohtani, Y. Sawada, Effects of grapefruit juice and orange juice components on P-glycoprotein- and MRP2-mediated drug efflux, Br. J. Pharmacol. 143 (7) (2004) 856–864. [25] H. Ohtani, T. Ikegawa, Y. Honda, N. Kohyama, S. Morimoto, Y. Shoyama, M. Juichi, M. Naito, T. Tsuruo, Y. Sawada, Effects of various methoxyflavones on vincristine uptake and multidrug resistance to vincristine in P-gp-overexpressing K562/ADM cells, Pharm. Res. 24 (10) (2007) 1936–1943. [26] W.D. Patino, O.Y. Mian, J.G. Kang, S. Matoba, L.D. Bartlett, B. Holbrook, H.H. Trout 3rd, L. Kozloff, P.M. Hwang, Circulating transcriptome reveals markers of atherosclerosis, Proc. Natl. Acad. Sci. U. S. A. 102 (9) (2005) 3423–3428. [27] T.C. Chou, Drug combination studies and their synergy quantification using the Chou-Talalay method, Cancer Res. 70 (2) (2010) 440–446. [28] T.C. Chou, M. Hayball, C.W. Lamble, Calcusyn-windows Software for Dose-Effect Analysis and Synergism/Antagonism Quantification, and User’s Manual, Biosoft, Cambridge, U.K, 1996–2007 www.biosoft.com. [29] R.B. van Breemen, Y. Li, Caco-2 cell permeability assays to measure drug absorption, Expert Opin. Drug Metab. Toxicol. 1 (2) (2005) 175–185. [30] P.R. Ding, A.K. Tiwari, S. Ohnuma, J.W.K.K. Lee, X. An, C.L. Dai, Q.S. Lu, S. Singh, D.H. Yang, T.T. Talele, S.V. Ambudkar, Z.S. Chen, The phosphodiesterase-5 inhibitor vardenafil is a potent inhibitor of ABCB1/P-glycoprotein transporter, PLoS One 6 (4) (2011). [31] Y.J. Wang, R.J. Kathawala, Y.K. Zhang, A. Patel, P. Kumar, S. Shukla, K.L. Fung, S.V. Ambudkar, T.T. Talele, Z.S. Chen, Motesanib (AMG706), a potent multikinase inhibitor, antagonizes multidrug resistance by inhibiting the efflux activity of the ABCB1, Biochem. Pharmacol. 90 (4) (2014) 367–378. [32] S.G. Aller, J. Yu, A. Ward, Y. Weng, S. Chittaboina, R. Zhuo, P.M. Harrell, Y.T. Trinh, Q. Zhang, I.L. Urbatsch, G. Chang, Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding, Science 323 (5922) (2009) 1718–1722. [33] K.M.R. Srivalli, P.K. Lakshmi, Overview of P-glycoprotein inhibitors: a rational outlook, Braz. J. Pharm. Sci. 48 (3) (2012) 353–367. [34] B.S. Vinod, T.T. Maliekal, R.J. Anto, Phytochemicals as chemosensitizers: from molecular mechanism to clinical significance, Antioxid. Redox Signal. 18 (11) (2013) 1307–1348. [35] O. Wesolowska, J. Wisniewski, K. Sroda-Pomianek, A. Bielawska-Pohl, M. Paprocka, D. Dus, N. Duarte, M.J.U. Ferreira, K. Michalak, Multidrug resistance reversal and apoptosis induction in human colon cancer cells by some flavonoids present in citrus plants, J. Nat. Prod. 75 (11) (2012) 1896–1902. [36] M. Evans, P. Sharma, N. Guthrie, Bioavailability of citrus polymethoxylated flavones and their biological role in metabolic syndrome and hyperlipidemia, in: Ayman Noreddin (Ed.), Readings in Advanced Pharmacokinetics—Theory, Methods and Applications, InTech, 2012. [37] J.A. Manthey, T.B. Cesar, E. Jackson, S. Mertens-Talcott, Pharmacokinetic study of nobiletin and tangeretin in rat serum by high-performance liquid chromatography-electrospray ionization-mass spectrometry, J. Agric. Food Chem. 59 (1) (2011) 145–151. [38] P. Giannakakou, R. Robey, T. Fojo, M.V. Blagosklonny, Low concentrations of paclitaxel induce cell type-dependent p53, p21 and G1/G2 arrest instead of mitotic arrest: molecular determinants of paclitaxel-induced cytotoxicity, Oncogene 20 (29) (2001) 3806–3813. [39] W. Ma, S. Feng, X. Yao, Z. Yuan, L. Liu, Y. Xie, Nobiletin enhances the efficacy of chemotherapeutic agents in ABCB1 overexpression cancer cells, Sci. Rep. 5 (2015) 18789. [40] G. Housman, S. Byler, S. Heerboth, K. Lapinska, M. Longacre, N. Snyder, S. Sarkar, Drug resistance in cancer: an overview, Cancers (Basel) 6 (3) (2014) 1769–1792. [41] Z. Liu, G. Zhu, R.H. Getzenberg, R.W. Veltri, The upregulation of PI3K/Akt and MAP kinase pathways is associated with resistance of microtubule-targeting drugs in prostate cancer, J. Cell. Biochem. 116 (7) (2015) 1341–1349. [42] H.A. Burris 3rd, Overcoming acquired resistance to anticancer therapy: focus on the PI3K/AKT/mTOR pathway, Cancer Chemother. Pharmacol. 71 (4) (2013) 829–842. [43] Z.W. Wang, Y.J. Huang, J.Q. Zhang, Molecularly targeting the PI3K-Akt-mTOR pathway can sensitize cancer cells to radiotherapy and chemotherapy, Cell. Mol. Biol. Lett. 19 (2) (2014) 233–242. [44] B.W. Vanhoecke, F. Delporte, E. Van Braeckel, A. Heyerick, H.T. Depypere, M. Nuytinck, D. De Keukeleire, M.E. Bracke, A safety study of oral tangeretin and xanthohumol administration to laboratory mice, In Vivo 19 (1) (2005) 103–107.

Please cite this article in press as: S.-L. Feng, et al., Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function, Pharmacol Res (2016), http://dx.doi.org/10.1016/j.phrs.2016.04.003