CCL5 protein was purchased from R&D systems, Inc. (Minneapolis, MN, USA; cat. no. 278-RN-050), Rabbit anti-mouse immunoglobulin G (IgG) H&L horseradish peroxidase (HRP; cat. no. ab6728; 1:5,000), goat anti-rabbit IgG H&L HRP (cat. no. ab6721; 1:5,000), goat anti-mouse IgG H&L (Alexa Fluor^® 594; cat. no. ab150116; 1:5,000) CCL5 (cat. no. ab56709; 1:1,000), phosphorylated (p-)CCR5 (cat. no. ab63411; 1:1,000), and CCR5 (cat. no. ab65850; 1:1,000) antibodies were purchased from Abcam (Cambridge, UK), p-AKT (cat. no. 4060; 1:2,000), AKT (cat. no. 9272), p-extracellular signal-regulated kinase (Erk) (cat. no. 4370; 1:1,000), Erk (cat. no. 4695; 1:1,000), matrix metalloproteinase (MMP)-2 (cat. no. 87809; 1:1,000), MMP-9 (cat. no. 13667; 1:1,000), E-cadherin (cat. no. 14472; 1:1,000) and vimentin (cat. no. 5741; 1:1,000) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

MDA-MB-231 and MDA-MB-453 TNBC cells, purchased from the Cell Bank of the Chinese Science Institute (Shanghai, China) were cultured in 1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin-streptomycin and maintained at 37°C and 5% CO₂. Human adipocytes were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.).

Human adipose tissue samples were obtained from mammary adipose tissue biopsies of healthy women (n=12; age 25–63 years; body mass index 24.2–29.0) undergoing surgical mammary reduction. All women were otherwise healthy and free of metabolic or endocrine diseases. Informed consent was obtained from every patient prior to the surgical procedure. The Ethics Committee of Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Shanghai, China) approved the present study. The adipose tissue was digested with collagenase, and adipose-derived stromal vascular fraction (SVF) cells were isolated and differentiated as previously reported (35). Informed consent was obtained from the patients for participation in the study.

An MTT assay was used to detect the effect of human adipocytes on TNBC cell viability. The supernatants of the SVF cells or adipocytes were collected, and MDA-MB-231 or MDA-MB-453 cells were incubated with the supernatant in 96-well plates at a density of 5,000 cells per well. After 24 h, epirubicin at concentrations of 0.01, 0.1, 0.5, 1 or 10 μM or emodin at concentrations of 1, 10, 50, 100 or 200 μM were added to the cell supernatant at 37°C, using three wells per concentration. After 24 h, MTT solution (10 μl) at a concentration of 5 mg/ml was added to 90 μl of culture media and incubated for 4 h at 37°C in a humidified atmosphere of 5% CO_2. The supernatant was removed and 100 μl per well of DMSO was added to the plates. The optical density was measured at 492 nm in a microplate reader. The experiment was performed in triplicate.

The invasive capacity of the cells was evaluated with a Transwell assay in 24-well plates (Corning Life Sciences, Corning, NY, USA) according to the manufacturer’s protocol. Briefly, the upper chambers were coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The MDA-MB-231 or MDA-MB-453 cells were seeded in the upper chamber of the Matrigel-coated Transwell culture system in 200 μl serum free-medium, and human SVF cells or adipocytes were seeded in the lower chamber in 500 μl medium with 10% FBS. The cells were then allowed to invade the Matrigel matrix for 24 h. Subsequently, the transmigrated cells were fixed and stained with crystal violet and counted in five randomly selected microscopic fields using an IX71 light microscope (Olympus Corporation, Tokyo, Japan). All experiments were performed in triplicate.

The MDA-MB-231 or MDA-MB-453 cells were seeded in the lower chamber of a Transwell culture system, with human SVF cells or adipocytes in the upper chamber. When cellular confluence reached ~90%, wounds were created in the confluent cells using 10-μl pipette tips. The cells were then rinsed with medium to remove any free-floating cells and debris. Wound healing was observed at 12 and 24 h within the scrape line, and representative images of the scrape lines were captured.

The levels of interleukin-8 (IL-8), insulin-like growth factor-1 (IGF-1) and CCL5 in the supernatant of the SVF cells, adipocytes, MDA-MB-231 and MDA-MB-453 cells were detected by ELISA according to the manufacturer’s protocol (RayBiotech, Norcross, GA, USA). The TNBC cells co-cultured with human adipocytes were treated for 24 h with 0.50 μM epirubicin and 50 μM emodin (for MDA-MB-231) or with 0.25 μM epirubicin and 25 μM emodin (for MDA-MB-453). The levels of CCL5 in the supernatant of the SVF cells, adipocytes, and co-cultures were detected using ELISA.

The MDA-MB-231 cells were seeded in the lower chamber of a Transwell culture system, with human SVF cells or adipocytes in the upper chamber. When cellular confluence reached ~90%, the MDA-MB-231 cells were treated with 6 μg/ml CCL5 antibody (cat. no. 2988; Cell Signaling Technology, Inc.), 100 pg/ml CCL5 (cat. no. 96-300-06; PeproTech, Inc., Rocky Hill, NJ, USA), and 0.50 μM epirubicin or 50 μM emodin for 24 h at 37°C and 5% CO₂. The protein lysates were obtained from the MDA-MB-231 cells treated with a lysis buffer (cat. no. 78501; Thermo Fisher Scientific, Inc.) with a protease inhibitor cocktail. The proteins were quantified using a BCA Kit (cat. no. 23225; Thermo Fisher Scientific, Inc.) and 10 μg protein was loaded and separated by 8–12% SDS-PAGE according to the protein weight and blotted onto a PVDF nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membrane was blocked in 5% milk in PBST for 1 h and then probed with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 h at room temperature. The signal was then visualized with enhanced chemiluminescence reagent, following the manufacturer’s protocol (cat. no. 34080; Thermo Fisher Scientific, Inc.).

All mouse experiments were performed in accordance with approved protocols from the Shanghai Medical Experimental Animal Care Commission (Shanghai, China). A total of 72 female Balb/C nude mice (age, 4–6 weeks; weight, 18–21 g) were obtained from Shanghai BiKai Bio-Technology (Shanghai, China) and housed under specific pathogen-free conditions at constant temperature (22±2°C) and humidity (55±5%) in a 12 h light/dark schedule with ad libitum access to water and standard laboratory chow. These mice were divided into six groups. Of the mice, 36 were fed with a basic diet and 36 received a high fat and sugar diet for 20 days. All mice were inoculated subcutaneously with MDA-MB-231 cells (1×10⁷) in 100 μl of 1640-Matrigel mixture (1:1 ratio) in the thoracic fat pad. When the tumor volumes reached an average of ~100 mm³, the mice were treated with saline orally (p.o.), three times per week, 40 mg/ml emodin p.o., once a day for 21 days, or 5 mg/ml epirubicin intravenously (i.v.) weekly for 3 weeks. The tumor volumes were measured every 5 days in two dimensions with vernier calipers. The tumor volumes were calculated using the following formula: Length × width² × 0.5. There were 12 mice were in each group, 6 of the 12 tumor samples were immersed in 4% paraformaldehyde for hematoxylin and eosin (H&E) staining, and 6 of 12 tumor samples were separated from liver and lung tissues and weighed. Tumor tissues from the in vivo experiments were collected for immunohistochemical analysis. The experiments were approved by the Ethics Committee of Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine.

The cells were cultured overnight on coverslips in 6-well plates. The coverslips were then removed and maintained in 4% polyformaldehyde for 15 min, and then washed three times in PBS solution. The coverslips were then treated with 0.2% Triton X-100 for 15 min, and then blocked with 3% goat serum (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 1 h. The coverslips were then incubated with E-cadherin antibody (1:1,000) at 4°C for 2 h, and then with secondary antibody linked with Alexa Fluor 594 (1:200) for 1 h. Following staining with DAPI, the cells on the coverslips were observed under a confocal microscope.

Tissues were immersed in 4% paraformaldehyde for 4 h, and then transferred to 70% ethanol. Individual lobes of the tissues biopsy materials were placed in processing cassettes, dehydrated through a serial alcohol gradient, and then stained with H&E. Following staining, the sections were dehydrated through increasing concentrations of ethanol and xylene.

All statistical analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). All data are presented as the mean ± standard deviation. Statistical significance was determined using a paired or unpaired Student’s t-test in cases of standardized expression data. One-way analysis of variance was performed for multiple group comparisons, and comparisons between two groups were made using the least significant difference method. P<0.05 was considered to indicate a statistically significant difference.

The present study investigated the effects of epirubicin and emodin on TNBCs in the supernatant of SVF cells or adipocytes. The MTT assay results suggested that the IC₅₀ values of epirubicin and emodin against MDA-MB-231 were 0.55 and 55.2 μM respectively, and when the MDA-MB-231 cells were incubated in the supernatant of adipocytes, the IC₅₀ values were 5.1 and 67.1 μM, respectively. Similar trends were observed with the MDA-MB-453 cells, demonstrating that, when co-cultured with adipocytes, the IC₅₀ of epirubicin was ~9.27 times higher, compared with the value obtained without adipocytes, whereas the IC₅₀ of emodin was only ~1.21 times higher, a finding which suggested that adipocytes led to resistance of TNBC to epirubicin, but not to emodin (Fig. 1A). The Transwell and wound healing assays demonstrated that adipocytes assisted in human TNBC MDA-MB-231 and MDA-MB-453 cell migration and invasion, and emodin caused more marked inhibition of TNBC cell migration and invasion, compared with epirubicin in the supernatant of adipocytes (Fig. 1B and C). Taken together, these findings showed that emodin inhibited TNBC cell proliferation and metastasis more efficiently than epirubicin in the supernatant of adipocytes.

It has been reported that the secretion of IL-8, CCL5 and IGF-1 by adipocytes can promote the migration of TNBC cells (16,17). In the present study, the results of the ELISA showed that the levels of IL-8, CCL5 and IGF-1 were higher in the cell supernatant of the adipocytes. The levels of these proteins were also measured in the cell culture supernatants following treatment with emodin or epirubicin. The results suggested that only CCL5 was downregulated following emodin treatment (Fig. 2A-F); however, no changes were observed following epirubicin treatment, indicating that emodin may inhibit TNBC proliferation and migration by decreasing the release of CCL5.

To investigate whether emodin inhibited TNBC cell invasiveness by antagonizing the secretion of CCL5 from adipocytes, TNBC cells were treated with 6 μg/ml CCL5 antibody, CCL5 100 pg/ml, and 0.5 μM epirubicin or 50 μM emodin for MDA-MB-231 cells, or 0.25 μM epirubicin or 25 μM emodin for MDA-MB-453 cells. The MTT assay results suggested that CCL5 antibody at a concentration of 6 μg/ml inhibited cell proliferation and that CCL5 reversed the inhibitory effect of emodin treatment (Fig. 3A). The Transwell assay suggested that CCL5 antibody inhibited cell invasion (Fig. 3B); however, CCL5 reversed the inhibitory effect of emodin treatment. The above results suggested that emodin may inhibit the proliferation and invasion of TNBCs by partially downregulating the secretion of CCL5.

To investigate the mechanism underlying the inhibitory effect of emodin on TNBC proliferation, migration and invasion via the CCL5/CCR5 pathway, the TNBC cells were treated with CCL5 antibody, emodin, or epirubicin, and the downstream signaling pathways of CCR5 and EMT-associated markers were analyzed by western blot or immunofluorescence assays. The results suggested that emodin inhibited the phosphorylation of AKT, activated GSK3β, downregulated the expression of β-catenin and mesenchymal-related proteins vimentin and snail, and upregulated the expression of epithelial-related protein E-cadherin. Epirubicin inhibited the phosphorylation of CCR5, promoted the phosphorylation of AKT, downregulated the expression of MMP-9 and snail, and upregulated the expression of GSK-β, β-catenin and E-cadherin. CCL-5 antibody downregulated the expression of CCL5, β-catenin, MMP-2, MMP-9 and snail, upregulated the expression of GSK3β and E-cadherin, and inhibited the phosphorylation of CCR5 and AKT. CCL5 promoted the phosphorylation of CCR5 and AKT, and upregulated the expression of CCL5, β-catenin, MMP-2, MMP-9 and snail. Emodin+CCL5 upregulated the expression of CCL5, activated GSK3b, MMP-2 and snail, and promoted the phosphorylation of CCR5 and AKT (Fig. 4A and B). These results indicated that emodin negatively regulated the downstream signaling pathways of CCR5 and EMT-associated markers, thereby inhibiting invasion and migration.

The in vivo assay showed that emodin inhibited the tumor growth and metastasis of TNBC cells by decreasing levels of CCL5 in the serum of mice fed a high fat and sugar diet. The nude mice were fed a high fat and sugar diet for 20 days and then inoculated with TNBC cells in the fat pad. After 7 days, the mice were treated with 40 mg/ml emodin p.o. once a day for 21 days, or 5 mg/ml epirubicin i.v. weekly for 3 weeks. The results suggested that emodin downregulated the level of CCL5, whereas epirubicin did not affect the level of CCL5 (Fig. 5A). Emodin had a similar effect on control nude mice bearing MDA-MB-231 with epirubicin; however, the efficacy of emodin was significantly higher on the nude mice fed a high fat and sugar diet than epirubicin according to the body weight (Fig. 5B), primary tumor (Fig. 5C), liver metastasis (Fig. 5D) and lung metastasis (Fig. 5E) tumor weight. The H&E staining data suggested that compared with the vehicle and epirubicin groups the emodin reduced lung (Fig. 5F) and liver metastasis (Fig. 5G) of the TNBC tumor. The above results indicated that emodin inhibited tumor growth and the lung and liver metastasis of TNBC cells by decreasing levels of CCL5 in mice fed a high fat and sugar diet (Fig. 5).