The human breast cancer cell lines MCF-7 and MDA-MB-231, and Madin-Darby Canine Kidney (MDCKII) cells were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). MDCKII cells are an epithelial cell line of canine kidney origin that are frequently used to investigate drug transporters in vitro, as they exhibit low expression levels of transporter proteins and low metabolic activity (32). MCF-7 and MDCKII cells were cultured in complete DMEM (Thermo Fisher Scientific Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). MDA-MB-231 cells were maintained in RPMI-1640 (Thermo Fisher Scientific Inc.) based culture medium supplemented with 10% FBS. The LZRS-IRES-eGFP plasmid expressing human ABCB4 was kindly donated by Professor Alfred Schinkel (Division of Molecular Oncology, The Netherlands Cancer Institute). For knockdown studies, the ABCB4 shRNA (5′-CUCAAUACGCGGCUAACAG-3′; Shanghai GenePharma Co., Ltd.) was cloned into the LZRS-IRES-eGFP vector. Doxorubicin-resistant breast cancer MCF-7/DOX and MDA-MB-231/DOX cells, MDCKII-patent (transfected with LZRS-IRES-eGFP-empty vector), MDCKII-ABCB4 (transfected with human LZRS-IRES-eGFP-ABCB4 expression vector), MCF-7/DOX and MDA-MB-231/DOX cells with ABCB4 knockdown (transfected with LZRS-IRES-eGFP-shABCB4) or overexpression (transfected with LZRS-IRES-eGFP-ABCB4 expression vector) were established in our laboratory as previously described (13). MCF-7/DOX and MDA-MB-231/DOX without transfection or treatment were used controls for ABCB4 knockdown/overexpression in the respective cell types. All the cell lines were maintained in an atmosphere containing 5% CO₂ at 37°C as previously reported (13).

The cytotoxicity of curcumin and the combination of curcumin (Sigma-Aldrich; Merck KGaA) and doxorubicin (Sigma-Aldrich; Merck KGaA) was assessed using the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies) assay. Cells were seeded in a 96-well plate at a density of 5×10³ cells/well. Cells were incubated with various concentrations of curcumin (0, 2.5, 5, 10, 20, 50 and 100 µM) for 72 h. For the doxorubicin chemosensitivity assay, cells were incubated with a combination of 10 µM curcumin and varying concentrations of doxorubicin (0, 2.5, 5, 10, 20, 50 and 100 µM) for 72 h. DMSO was used as the control. Following incubation, 10 µl CCK-8 solution was added to each well, and cells were incubated for an additional 4 h. The absorbance was measured at a wave length of 570 nm using a fluorescence spectrofluorometer (cat no. F-7000; Hitachi Ltd.). IC₅₀ values were calculated using the cell survival data curve.

The intracellular accumulation of doxorubicin in cells was measured using a flow cytometer. The cells were cultured in 6-well plates at a density of 1×10⁶ per well, and were cultured to 80% confluence with or without 10 µM curcumin treatment for 72 h. The cells were subsequently treated with 30 µM doxorubicin for 4 h, and washed three times with chilled PBS. The cell-associated mean fluorescence intensity of doxorubicin was detected using FACSCalibur (Beckman Coulter, Inc.) with excitation/emission wavelengths of 485/580 nm. The results were analyzed though CyExpert 2.0 (Beckman Coulter, Inc.) software.

Total RNA was extracted from the cells with TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The extracted RNA was reverse transcribed into cDNA using the Revert Aid™ H Minus First Strand cDNA Synthesis kit (Takara Bio, Inc.) according to the manufacturer's protocols. qPCR was performed using the SYBR^® Green PCR Master mix (Takara Bio, Inc.). The following primer pairs were used for the qPCR: ABCB4, forward 5′-TGGCCCTGGTTGGAAGTAGTG-3′ and reverse 5′-AGAAGGATCTTGGGGTTGCGAA-3′; and β-actin, forward 5′-GGATGCAGGAGATCACTG-3′ and reverse 5′-CGATCCACACGGAGTACTT-3′. qPCR was conducted as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Relative quantification of ABCB4 mRNA was analyzed using the 2^−ΔΔCq method relative to the β-actin expression level in each sample (33).

Total protein was extracted using radioimmunoprecipitation protein lysis buffer (Beyotime Institute of Biotechnology) and quantified using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Protein extracts (50 µg/lane) were separated by via SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). The membranes were blocked for 2 h at room temperature with 5% non-fat milk. Subsequently, the membranes were incubated with primary antibodies against ABCB4 (1:1,000; cat. no. ab24108, Abcam) or β-actin (1:5,000; cat. no. A5441, Sigma-Aldrich; Merck KGaA) at 4°C overnight. The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; cat. no. ab97023, Abcam) for 2 h at room temperature. Protein bands were visualized using the ECL™ Prime (GE Healthcare) and a LAS-3000 imager (Fujifilm).

Cellular transport assay was performed as described previously (13). Briefly, MDCKII-parent (transfected with LZRS-IRES-eGFP-empty vector) and MDCKII-ABCB4 (transfected with LZRS-IRES-eGFP-ABCB4 expression vector) cells (1×10⁶/insert) were seeded onto microporous polycarbonate membrane inserts (pore size, 3 µm; diameter, 24 mm; Costar; Corning, Inc.). The plates were incubated at 37°C with 5% CO₂ for 3 days and subsequently used for assays. Prior to experimentation, cells were cultured in Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc.) with or without 100 µM verapamil (Sigma-Aldrich; Merck KGaA) or 10 µM curcumin for 2 h. Opti-MEM was subsequently replaced with fresh DMEM supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.) and 100 nM (³H) doxorubicin with or without 100 µM verapamil or 10 µM curcumin. The radioactivity of transported (³H) doxorubicin was determined using a liquid scintillation counter (cat no. LS6500; Beckman Coulter, Inc.). The effect of curcumin on ABCB4-mediated doxorubicin transport was calculated as the fraction of doxorubicin recovered in the acceptor compartment vs. the fraction added in the donor compartment at the beginning of the experiment.

ATPase activity was assayed to determine the functionality of the ABC transporter. MDCKII-ABCB4 cells (1×10⁶) were incubated at 37°C for 30 min in the presence of sodium orthovanadate (0.3 mM) in ATPase assay buffer (50 mM KCl, 5 mM sodium azide, 2 mM EDTA, 10 mM MgCl₂ and 1 mM dithiothreitol; pH 6.8) to measure the amount of inorganic phosphate that was released. Membrane proteins were isolated from cells using a Membrane and Cytosol Protein Extraction kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. A total of 30 µg membrane extracts were incubated with increasing concentration of curcumin (0, 5, 10, 15 and 20 µM) in the presence and absence of 5 µM verapamil. The reaction was initiated by the addition of 5 mM ATP and incubated for 20 min at 37°C. The reaction was terminated by the addition of 0.1 ml 5% SDS solution, and the inorganic phosphate released was quantified with a colorimetric reaction at 340 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

Statistical analysis was performed using SPSS software (version 20; IBM Corp.). All values are presented as the mean ± SEM. One-way ANOVA and Tukey's post hoc test were performed to analyze the differences between the different groups. P<0.05 was considered to indicate a statistically significant difference.

Prior to testing the reversal effects of curcumin, its cytotoxicity was evaluated using the CCK-8 assay. Curcumin (20 µM) exhibited moderate cytotoxic effects on parental and resistant cells following 72 h of treatment (Fig. 1). The majority (>90%) of parental and doxorubicin-resistant cells survived at a concentration of 10 µM curcumin, suggesting that curcumin is safe to be used up to a concentration of 10 µM. Thus, 10 µM curcumin was used for the subsequent assays using MCF-7/DOX and MDA-MB-231/DOX cells.

Preliminary results revealed that ABCB4 was overexpressed in MCF-7/DOX and MDA-MB-231/DOX cells, and that it results in drug resistance (12). The present study investigated the role of ABCB4 in the effects of curcumin on doxorubicin resistance. The effect of curcumin on doxorubicin cytotoxicity in control the group, ABCB4 knockdown and ABCB4 overexpression in MCF-7/DOX and MDA-MB-231/DOX cells was investigated. As presented in Table I, compared to the DMSO-treated cells, curcumin reduced the IC₅₀ value of doxorubicin in all the analyzed cells. The most significant reduction was observed in ABCB4 overexpressing cells, while the least significant reduction was found in ABCB4 knockdown cells. The intracellular accumulation of doxorubicin in these cells was subsequently detected in the presence or absence of curcumin. As expected, compared with DMSO-treated group, co-incubation of doxorubicin and curcumin resulted in a significant increase in intracellular concentration of doxorubicin, and the rate of increase was proportional to the level of ABCB4 expression (Fig. 2).

The reversal of ABCB4-mediated doxorubicin resistance may occur by ABCB4 down regulation or inhibition of the function of ABCB4. Therefore, the effect of curcumin on the expression level of ABCB4 following treatment of the doxorubicin-resistant cell lines MCF-7/DOX and MDA-MB-231/DOX with 10 µM curcumin for 0, 24, 48 and 72 h was investigated. The results revealed that following treatment with curcumin, the mRNA and protein levels of ABCB4 in the two cell lines were not significantly altered (Fig. 3). Therefore, curcumin may inhibit the transport function of ABCB4 to reduce doxorubicin resistance.

In order to clarify whether the reversal of doxorubicin resistance by curcumin occurs due to inhibition of the efflux function of ABCB4, an efflux assay was performed. The amount of (³H)-doxorubicin that was pumped into the extracellular medium was increased in MDCKII-ABCB4 cells compared with MDCKII-parent cells (Fig. 4). Incubation with curcumin significantly reduced the efflux of (³H)-doxorubicin from MDCKII-ABCB4 cells. Verapamil is an inhibitor of ABCB4 function and was used as control. Verapamil decreased the efflux of (³H)-doxorubicin in MDCKII-ABCB4 cells. Curcumin and verapamil did not alter the rate of efflux of (³H)-doxorubicin in the MDCKII-parent cells.

To assess whether curcumin interacts directly with ABCB4, the effect of curcumin on ABCB4 ATPase activity was investigated in crude membranes expressing ABCB4. The results revealed that curcumin slightly stimulated basal ATP hydrolysis by ABCB4 at low concentrations (0.25–1 µM), but inhibited ATPase activity at higher concentrations. In addition, curcumin inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner (Fig. 5).