The human breast cancer cell line MCF-7 and resistant derivative cell line MCF-7/Adr were gifted by the Department of Surgery, Wuhan Union Hospital, Hubei, China. Sensitive human breast cancer MCF-7/s cells were obtained from ATCC (USA). The MCF-7/adr cells (Michigan Cancer Foundation-7/adriamycin resistant) were kindly provided by Dr. Huang Tao (University of Science and Technology of Xiehe Hospital at Huazhong Surgery Laboratory Center, Wuhan, Hubei). The cell line is derived from the parental drug-sensitive MCF-7 cells by stepwise selection with Dox. To maintain the drug-resistant phenotype, the cells were cultured in the presence of 1 μg/ml Dox in RPMI-1640 culture medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 10 mM glutamine, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). Cells were maintained at 37ºC in 5% CO₂/95% air (20–22).

Genistein and rhodamine (Rh123) were obtained from Sigma-Aldrich (USA). Doxorubicin hydrochloride was purchased from Shanghai Biological Engineering Co., Ltd. (China). Oligo(dt) was synthesized by Shanghai Biological Engineering Co. Taq PCR MasterMix was purchased from Tiangen (China). Primers were synthesized by Shanghai Biological Engineering Co. Anti-human permeability glycoprotein (P-gp) monoclonal antibody was purchased from Abcam (USA). Anti-c-erbB2 [human epidermal growth factor receptor 2 (Her2/Neu)] polyclonal antibody was purchased from Dako Corp. (USA). Anti-mouse IgG-horseradish peroxidase (HRP) and anti-rabbit IgG-HRP were purchased from Beijing Ding National Biotechnology Co. (China). A Coulter DNA PREP™ reagents kit was purchased from Beckman-Coulter (USA). An Annexin V-FITC apoptosis detection kit was purchased from Shanghai Yan-Bin Chemical Technology (China). The F-7000 fluorescence spectrometer was purchased from Hitachi (Japan), and the Hitachi UV spectrophotometer was from Eppendorf (Germany).

Cells were cultured in RPMI-1640 culture medium with 10% fetal calf serum at 37ºC with 5% CO₂. Cells were trypsinized with 0.25% trypsin and passed every 2 or 3 days. MCF-7/Adr cells were maintained in medium supplemented with doxorubicin (1.0 μg/ml) for two weeks. Cells were diluted from 5×10⁴ to 5×10⁵/ml for use in the experiments.

The purity of genistein was greater than 98%. Twenty-five milligrams of genistein was dissolved in 1 ml dimethylsulfoxide (DMSO) and stored at 4°C. Before use, genistein was diluted in culture medium to the required concentration, with the final percentage of DMSO <0.2%. Twenty-five milligrams of doxorubicin was dissolved in 10 ml saline and stored at 20°C. For experiments, doxorubicin was serially diluted to final concentrations of 0.1, 1, 10 and 100 μM.

MCF-7/Adr cells in the logarithmic growth phase were seeded in 96-well culture plates. To each well, we added 30 μmol/l genistein and 0.07, 0.7, 7 or 70 μM doxorubicin, in a final volume of 200 μl (n=8 for each concentration). Wells with medium only (no cells) were used to measure the background, and seeded wells given untreated medium served as the controls. Forty-eight hours after treatment, MTT was added to measure proliferation. Q-value was calculated by the formula of Guinness (17–19). IC₅₀ and the reversal fold were also calculated. The reversal fold = IC₅₀ (chemotherapy drugs)/IC₅₀ (genistein).

Quantification of doxorubicin concentrations was carried out using a standard curve. The standard curve was established using standard solutions of different doxorubicin concentrations (0, 0.01, 0.05, 0.25, 1.25, 6.25 and 31.25 μM). Cell culture medium was added to yield final genistein concentrations of 7, 15, 30, 60 and 120 μmol/l, and 0.7, 7 and 70 μM doxorubicin. After experimental culture, the supernatants were collected and centrifuged, and fluorescence intensity was measured. The concentration of doxorubicin in the supernatant was calculated according to the standard curve.

MCF-7/Adr cells in the logarithmic growth phase were seeded in 24-well plates. The cells were divided into following groups: control group (culture medium), doxorubicin groups (10 or 100 μM), genistein groups (30 or 60 μmol/l), low-dose combination group (1 μM doxorubicin + 30 μmol/l genistein), and high-dose combination group (10 μM doxorubicin + 60 μmol/l genistein). The final concentration of DMSO was 0.1%. Then cells were harvested and washed twice with cold PBS. Cells were suspended in cold 70% ethanol and fixed overnight at -20°C. Fixed cells were centrifuged, resuspended in resuspension buffer, and 100 μl propidium iodide (PI; 50 μg/ml) was added and incubated for 30 min at 4°C for staining. A 200-μl sample was transferred to an injection tube, along with 100 μl Coulter DNA-PREP reagents kit. After 2 min of mixing, 1 ml Coulter DNA-PREP reagents kit stain was added and the solution was mixed. PI single staining was used to assess cell cycle via flow cytometry. PI and Annexin V-FITC double staining was used to measure apoptosis. To measure Rh123, a fluorescent substrate of P-gp, a cell suspension was added to 2 mg/l Rh123 or Rh123 + different concentrations of genistein and incubated at 37°C for 45 min. Cells were washed twice with cold PBS, and Rh123 concentrations were measured by flow cytometry.

MCF-7/Adr cells in the logarithmic growth phase were seeded in 6-well plates and divided into the following groups: control, doxorubicin (1 or 10 μM), genistein (15, 30 or 60 μmol/l). After 48 h of treatment, cells were harvested and total-RNA was extracted. The A260:A280 ratio was ≥1.8. RT-PCR was performed according to the two-step RT-PCR amplification kit instructions. The primers were as follows: mdr-1 forward, 5′-CCCATCATTGCAATAGCAGG-3′ and reverse, 5′-GTTCAAACTTCTGCTCCTCA-3′; Her2/neu forward, 5′-GCCCTCATCCACCATAACACC-3′ and reverse, 5′-CATTCCTCCACGCACTCCTG-3′; β-actin forward, 5′-AGCGAGCATCCCCCAAAGTT-3′ and reverse, 5′-GGG CACGAAGGCTCATCATT-3′. The PCR product of mdr-1 is 157 bp; the PCR product of Her2/neu is 220 bp; and the PCR product of control β-actin is 200 bp. PCR conditions were as follows: 94°C denaturation for 30 sec, 57°C refolding for 30 sec, and 72°C extension for 30 sec, for a total of 30 cycles.

Cells were washed with cold PBS and lysed with protein lysis buffer for 30 min. Cell lysates were centrifuged at 12,000 × g at 4°C for 15 min. After centrifugation, supernatants were considered as total protein extract. The total amount of protein for loading was adjusted using the measured protein concentration. Six microliters of 5X sample buffer was added to each sample for a final volume of 30 μl. Samples were boiled at 100°C for 5 min (protein denaturation) and subjected to electrophoresis at 4°C. Proteins in the gel were transferred to a polyvinylidene fluoride membrane and blotted with anti-human P-gp antibody (diluted 1:20 in blocking solution) and the anti-c-erbB2 antibody (1:500) overnight at 4°C. HRP-conjugated secondary antibodies were added for 2 h.

SPSS 17.0 software was used for variance and regression analyses and pairwise comparisons Q-test. Data are presented as means ± standard deviation (SD). Comparison between groups was performed via t-test. P<0.05 was considered to indicate a statistically significant difference.

It is known that MCF-7/Adr cells are resistant to doxorubicin once the cell line is established. To confirm that MCF-7/Adr cells do have this biological feature, we compared the sensitivity of MCF-7/S and MCF-7/Adr cells. As shown in Fig. 1, doxorubicin inhibited the cell proliferation of MCF-7/S cells in a dose- and time-dependent manner; however, doxorubicin had no inhibitory effect on MCF-7/Adr cells. In MCF-7/Adr cells, the IC₅₀ for a 48-h treatment was 117.15 μM (4.75±0.04 to 556.21±51.35 μM), whereas the IC₅₀ for a 72-h treatment was 54.42 μM (8.95±0.08 to 486.90±45.33 μM). Taken together, our results confirmed that the MCF-7/Adr cells were resistant to doxorubicin.

Several studies have shown that genistein can enhance the cytotoxic effects of doxorubicin and reduce the chemoresistance of tumor cells (17–19). To test this observation, we treated MCF-7/Adr cells with genistein (30 μmol/l) along with increasing doses of doxorubicin. We found that genistein had a synergistic effect with doxorubicin at all tested doses (Q>1.15; Table I). The inhibitory effect of doxorubicin and genistein was significantly greater than that of the control and doxorubicin alone (P<0.01; Table I).

Our results revealed that genistein enhanced the cytotoxic effect of doxorubicin. To better understand the molecular mechanism of this observation, we investigated whether genistein increases the intracellular accumulation of doxorubicin in MCF-7/Adr cells. The intracellular accumulation of doxorubicin was estimated by the formula y = 152.55 + 99.496x, R²=0.954. As presented in Table II, genistein significantly increased the intracellular accumulation of doxorubicin (P<0.01). In the MCF-7/Adr cells, increased concentration of doxorubicin alone did not significantly upregulate the intracellular accumulation of doxorubicin, and the maximal intracellular loading of doxorubicin was extremely limited in the doxorubicin alone group (Table II). However, genistein treatment significantly increased the intracellular accumulation of doxorubicin in a dose-dependent manner. More importantly, the maximal loading of doxorubicin was greatly increased with genistein treatment (Table II).

To further understand the mechanism underlying doxorubicin accumulation in MCF-7/Adr cells, we tested the effect of genistein on the intracellular accumulation of Rh123, a fluorescent substrate of P-gp. Rh123 is transported by P-gp and therefore can be used as a measure of drug extrusion by the plasma membrane (17–19). The intracellular accumulation assay showed that genistein had no effect on the intracellular accumulation of Rh123 (Fig. 2). Assays of Rh123 excretion by MCF7/S and MCF7/Adr cells also showed that genistein had no effect on the excretion of Rh123 (Fig. 2). These results suggest that genistein specifically affects the intracellular accumulation of doxorubicin but not that of Rh123.

Doxorubicin intercalates into DNA and induces cell cycle arrest. We tested whether genistein increases doxorubicin-induced cell cycle arrest. Compared to the control, both genistein and doxorubicin significantly induced MCF-7/Adr cell cycle arrest at the G2/M phase (P<0.01; Table III and Fig. 3). Genistein treatment at 60 μmol/l for 48 h and 72 h resulted in decreased percentages of cells in the G1 and G2/M phases but increased the percentages of cells in the S phase. The effect of combined genistein and doxorubicin treatment on cell cycle arrest was much stronger than that of either genistein or doxorubicin alone (Table III and Fig. 3).

We tested whether genistein enhances doxorubicin-induced apoptosis. Doxorubicin alone did not cause obvious apoptosis in the MCF-7/Adr cells. However, when treated with genistein alone or in combination with doxorubicin, MCF-7/Adr cells became apoptotic (P<0.01; Table IV and Fig. 4). The genistein and doxorubicin combination group showed the highest percentage of apoptotic cells (Table IV and Fig. 4).

To better understand the molecular mechanism of genistein’s effects, we tested the influence of genistein on the expression of mdr-1 and Her2/neu mRNA. Genistein and doxorubicin alone or in combination had no effect on mdr-1 mRNA expression (P>0.05; Fig. 5A). However, genistein downregulated Her2/neu mRNA expression in a dose-dependent manner (P<0.01; Fig. 5A). The combination of genistein and doxorubicin also significantly suppressed Her2/neu mRNA expression (P<0.01; Fig. 5A).

We examined the expression of P-gp and c-erbB2 proteins. Genistein and doxorubicin alone or in combination had no effect on the expression of P-gp (P>0.05; Fig. 5B). However, genistein downregulated the expression of c-erbB2 (P<0.01; Fig. 5B).