Monoclonal antibodies against vascular endothelial growth factor (VEGF; sc-7269), caspase-3 (sc-7148), ERα (sc-73562), progesterone receptor (PR; sc-130071), Bax (sc-20067), Bcl-2 (sc-130308), cyclin D1 (sc-70899) and NF-κB p65 (sc-372) were provided by Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). RPMI-1640 medium (Gibco-Invitrogen, Grand Island, NY, USA) was purchased from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (Grand Island, NY, USA).

Pulmonary adenocarcinoma cells (A539), human gastric carcinoma cells (MKN-45), hepatocellular carcinoma cells (HepG2), human colon cancer cells (SW620), human umbilical vein endothelial cells (HUVECs) and human breast cancer cells [MCF-7 (ER⁺), MDA-MB-231 (ER⁻)] were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

The cells were cultured in complete Dulbecco's modified Eagle's medium (DMEM) medium containing 10% fetal bovine serum (FBS). The cells were cultured in 24-well plates at the concentration of 40,000 cells/ml. The pigment compound VI (benzoquinone) was diluted in acid-ethanol (the control groups were treated with an equal volume of acid-ethanol solution). The cells were then incubated at 37°C in a humidified atmosphere of 5% CO₂ for 24 h. The cells were treated with 17β-estradiol (E₂, 0.1 µM; Sigma-Aldrich, Castle Hill, NSW, Australia), fulvestrant (Fu, 0.13 µM; Sigma, Auckland, New Zealand), compound VI (5, 10 and 15 µM), or the corresponding control solutions. The cells were incubated at 37°C, 5% CO₂ in a humidified incubator for 24 h. Assays were initiated by the addition of 100 µl MTT solution (2 mg/ml) to each well and incubating the cells for an additional 4 h at 37°C. Subsequently, the medium was removed and 1 ml dimethylsulphoxide (DMSO) was added to each well. Finally, the supernatants were transferred to 96-well plates in triplicate, which were read at a wavelength of 490 nm with a Thermo Scientific Multiskan^® Spectrum spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The pigment inhibitory concentration (IC₅₀, defined as the drug concentration at which cell growth was inhibited by 50%) was assessed from the dose-response curves. The percentage viability of the pigment extract was calculated using the formula: cell viability (%) = 100 − [(OD_{pigment control}) − (OD_pigment)/(OD_pigment)] ×100.

Cell behavior was monitored in real-time using the xCELLigence system E-Plate (ACEA Biosciences Inc., San Diego, CA, USA). A total of 20,000 MCF-7 cells was seeded in each well and fresh DMEM was added for a duration of 24 h. The DMEM was then removed from these wells and medium containing compound IV at various concentrations (VI IC₂₅, compound VI IC₂₅; VI IC₇₅, compound VI IC₇₅) was then added. The impedance value of each well was automatically monitored by the xCELLigence system for a duration of 72 h and expressed as a cell index (CI) value. The CI value was used to represent the cell status based on the measured frequency-dependent electrical impedance. The CI value was used as a global guide to cellular behaviour including attachment, proliferation and spreading.

The cells were seeded onto glass coverslips placed in a 24-well plate (5×10⁵ cells/well) in complete DMEM medium containing 10% FBS and incubated overnight. The cells were treated with VI IC₅₀, Fu or the respective controls or the vehicle. The VI IC₅₀ Con used was an equal volume of acid-ethanol solution. The fulvestrant control (Fu Con) used was an equal volume of acetate solution. Complete medium only was used as the vehicle. The cells were fixed using 4% paraformaldehyde, 0.15% picric acid in 1X phosphate-buffered saline (PBS) for 20 min at room temperature. The cells on glass coverslips were incubated with the indicated primary antibodies at room temperature for 1 h. After washing 3 times with PBS for 5 min, the slides were incubated for 15 min with the corresponding biotinylated secondary antibodies (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), followed by 30 min of incubation with streptavidin-horseradish peroxidase (DakoCytomation A/S, Glostrup, Denmark). Color was developed with 3′-diaminobenzidine (DAB) for 3–7 min. Counterstaining was performed with hematoxylin for 5 min, and the slides were coverslipped. To evaluate ERα and PR expression, a semi-quantitative method was used to determine the reaction intensity and percentage of positive cells (deep blue) from 10 representative fields of the specimen at ×100 magnification (Olympus DSX500, Olympus Corp., Tokyo, Japan).

This method was used to determine the expression of NF-κB p65, caspase-3 in MCF-7 cells. The cells were seeded onto glass cover-slips placed in a 24-well plate (5×10⁵ cells/well). The groups and treatments were as described above. The cells were treated with various concentrations of the pigment compound VI. Following treatment, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were blocked with 10% normal goat serum (Gibco) prior to incubation with rabbit anti-NF-κB p65, caspase-3 (Santa Cruz Biotechnology, Inc.) or negative control rabbit IgG (Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) overnight at 4°C. After rinsing with PBS, the cells were incubated with goat anti-rabbit Cy5 (Abcam, Cambridge, MA, USA) and 1 µg/ml of Hoechst 33342 (Sigma) for 1 h at room temperature. Immunolabelling was visualized and imaged using an Olympus DP71 fluorescence microscope (Olympus, Center Valley, PA, USA).

The cells were washed twice with cold PBS and lysed in ice-cold RIPA buffer (Sigma) supplemented with complete mini-protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). The cell lysates were centrifugated at 4°C, 12,000 rmp, for 30 min. We collected the supernatant and measured the protein concentration using a Bradford kit (Thermo Fisher Scientific, Inc.). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis was carried out using standard protocols with 15 µg of total protein/well. The primary antibodies used were as follows: mouse anti-human cyclin D1, VEGF, NF-κB p65, Bax and Bcl-2 monoclonal antibodies (1:200). Following incubation with the primary antibodies at 4°C overnight, the membranes were washed with PBST 3 times, and were then incubated with HRP-labeled secondary antibody (1:2,000; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.) for 2 h at room temperature. The blots were visualized using a chemiluminescent detection kit (Thermo Fisher Scientific, Inc.). The gray ratio of each band was measured using ImageJ software. Relative protein expression was calculated using the following gray ratio: gray ratio = gray of target band/β-actin gray.

SPSS 11.5 statistical software was used for data processing, and data are the means and standard deviation (means ± SD). Comparisons between 2 groups were made using the LSD test and Hochberg statistical methods for statistical processing. The value of α=0.05 was used as a significant test standard, with a value of P<0.05 used to indicate a statistically significant difference.

MTT assay was used to screen the isolated compound VI against a panel of human cancer cell lines, including A549, MCF-7, MKN-45, HepG2 and SW620 cells. As shown in Fig. 2, the isolated compound VI displayed broad spectrum anti-proliferative activities in a dose-dependent manner (Fig. 2A). Compared with the corresponding controls, compound VI inhibited the proliferation of different tumor cells in a dose-dependent manner (Fig. 2A–C), while it did not have any apparent effect on normal cells, such as HUVECs (Fig. 2D). We also found that compound VI exerted a more potent inhibitory and significant effect on MCF-7 cell proliferation (Fig. 2A–C).

To further explore the correlation between the concentration of compound VI and its inhibitory effect on MCF-7 cells, we employed 9 gradients of compound VI. The results revealed that 7 µM of compound VI exerted a 25% inhibition, while 11 µM of compound VI exerted a 50% inhibition (Fig. 2D). Our findings further validated that compound VI exerts an inhibitory effect on MCF-7 cell proliferation in a concentration-dependent manner.

The xCELLigence system is a label-free cell-based assay system, which integrates microelectronics and cell biology, and is suitable for the uninterrupted monitoring of the biological processes of living cells. The impedance measurements, which are displayed as CI values, provide quantitative information about the biological status of the cells including cell number, viability and morphology (13). In this study, we monitored the conditions of MCF-7 cells treated with compound VI at various concentrations using the real-time cell-based assay (RTCA) platform. The distinct patterns can be seen on the graph, which can be attributed to cell adhesion and cell proliferation (0–24 h) (Fig. 3).

After having determined the optimal conditions to study the behavior of the MCF-7 cells (data not shown), we then determined whether compound VI affects the behavior of MCF-7 cells. In order to accomplish this, the MCF-7 cells were seeded in DMEM containing various concentrations of compound VI. Cell behavior was monitored using RTCA over a period of 72 h. The results revealed that the CI value of MCF-7 cells was robust and did not exhibit any detectable difference within DMEM medium or without compound VI treatment from 0 to 24 h (Fig. 3). However, cell proliferation was significantly compromised with compound VI treatment from 24 to 72 h (Fig. 3). As shown in Fig. 3, the cells in the vehicle-treated group continuously grew more rapidly (dark purple line); however, cells in the treated group grew at a slower rate during the period of 24–48 h. The CI value of the treatment groups was the hightest at 46 h. After this time point, the CI values of the treated cells gradually decreased [VI IC₂₅ (red line), VI IC₅₀ (dark blue line), Fu (green line) and VI IC₇₅ (light purple line)], while the cells in the VI IC₅₀ control group grew more rapidly [Fig. 3, cyan (light blue) line]. Taken together, this observation suggests that compound VI exerts an inhibitory effect on MCF-7 cell proliferation, and these inhibitory effects are evident when cells are treated for a period >24 h.

ER signaling is the most attractive target for the clinical therapy of ER⁺ breast cancer. Within the anti-proliferative environment, there is compelling evidence to indicate that, in estrogen-sensitive human breast cancer cell lines, such as MCF-7, treatment with certain agents disrupts estrogen-responsive gene expression and inhibits estrogen-dependent proliferation. PR expression is traditionally used as a clinical indicator of ER function (that is, PR is an ER target gene) (14).

In this study, to determine whether the compound affects the expression of ERα and PR in MCF-7 cells, we employed immunohistochemisty staining assay. Compound VI was added at the IC₅₀ concentration to the MCF-7 cells for 24 h. The number of MCF-7 cells whose cytoplasm was stained deep blue represented the cells expressing ERα and PR. Immunohistochemical analysis demonstrated that the expression of ERα and PR was markedly inhibited in the cells treated with compound VI at the IC₅₀ concentration (compare Fig. 4A or B, VI IC₅₀ to Fig. 4, VI IC₅₀ Con).

The effects of the benzoquinone compound on cell viability were evaluated by MTT assay in ER⁺ MCF-7 and ER⁻ MDA-MB-231 cells (Fig. 1A). Compared to the controls, compound VI exerted an overall decrease in the OD value in a dose-dependent manner. The compound, however, had no effect on MDA-MB-231 cells, suggesting that the anti-proliferative effects of compound VI are dependent on the presence of ERα (Fig. 5).

Of note, the activation of the pro-inflammatory transcription factor, NF-κB, may play a key role in ER⁺ tumors, while caspases-3 plays an essential role in cell apoptosis and has been termed an 'executioner' protein for its role in cell apoptosis and may be used as a biomaker to predict tumor response to treatment (15). Quinone has been shown to induce the apoptosis of tumor cells by suppressing NF-κB, activating the Akt pathway and suppressing tumor angiogenesis (16). Thus, in this study, we wished to determine whether compound VI affects the expression of NF-κB and caspases-3. The results of immunofluorescence staining revealed that caspase-3 was exclusively localized in the cytoplasm of MCF-7 cells (Fig. 6A, vehicle) while NF-κB was observed in the nuclei of MCF-7 cells (Fig. 6B, vehicle). We further explored the changes in the expression of NF-κB and caspase-3 following treatment of the cells with compound VI (VI IC₂₅ and VI IC₅₀) for 24 h. We observed that the expression of NF-κB was decreased (Fig. 6B), whereas that of caspase-3 was increased in the MCF-7 cells following treatment with compound VI (Fig. 6A). To further evaluate NF-κB expression and cleaved caspase-3 activation, their protein levels were examined in the cells by western blot analysis (Fig. 7). Treatment with compound VI inhibited NF-κB expression and increased the cleavage of caspase-3.

To further explore the molecular mechanisms responsible for the inhibitory effects of compound VI on MCF-7 cell proliferation, we examined the protein expression of Bax, Bcl-2, NF-κB p65, caspase-3, VEGF and cyclin D1 by western blot analysis. The results revealed that following treatment with compound VI at IC₂₅ and IC₅₀ for 24 h, the levels of Bax and cleaved caspase-3 markedly increased compared to those of control group (p<0.01; Fig. 7). By contrast, the levels of Bcl-2, NF-κB, cyclin D1 and VEGF in the cells treated with compound VI at IC₅₀ for 24 h were significantly lower than those of the control group (p<0.01; Fig. 7). However, the levels of cleaved caspase-3 and Bax in the cells treated with compound VI at IC₅₀ were markedly higher than those of the control group (p<0.01; Fig. 7B).